Prominin-1 peptide fragments and uses thereof

ABSTRACT

Described herein are peptide compositions of a prominin-1, which have regenerative activity. As such the peptides are useful when regeneration is needed, for example, to enhance angiogenesis, increase VEGF binding to endothelial cells, promote vasodilation, enhance cell migration, enhance cell proliferation, stimulate neuronal growth, prevent neurodegeneration, and/or promote neuroregeneration.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of the InternationalApplication Serial No. PCT/US2009/051971, filed on Jul. 28, 2009, whichclaims benefit under 35 U.S.C. §119(e) of U.S. provisional application61/084,052 filed Jul. 28, 2008, both of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The field of the invention relates to the field of regeneration and themodulation of the biological effects of pro-angiogenic factors.

BACKGROUND OF INVENTION

Angiogenesis is the formation, development and growth of new bloodvessels. The normal regulation of angiogenesis is governed by a finebalance between factors that induce the formation of blood vessels andthose that halt or inhibit the process. When this balance is upset, itgenerally results in pathological angiogenesis. A great number ofpathologies arise from either an excess of angiogenesis or, conversely,an insufficient angiogenesis. Regulating angiogenesis with angiogenic(for insufficient angiogenesis) or angiostatic (for excessiveangiogenesis) factors is therefore of great therapeutic interest in anumber of medical fields such as ophthalmology, oncology anddermatology. Regulation of angiogenesis can provide approaches for thetreatment of vascular diseases, for example, diseases characterized bypoor capillarity and/or neurogenesis, including stroke, coronary arterydisease, peripheral muscle impairment associated with chronicobstructive pulmonary disease, wound healing, and Alzheimer's disease.

SUMMARY OF THE INVENTION

Embodiments of the present invention are based on the discoveries thatshort peptides derived from a penta span transmembrane glycoprotein havedistinct effects on angiogenesis. The inventors found that shortpeptides from prominin-1 (prom-1) bind VEGF (see table 1), an endogenouspro-angiogenesis factor that is important for normal growth anddevelopment but is also involved during unwanted and aberrantvascularization such as in cancer and diabetic retinopathy. In addition,the short peptides promoted VEGF binding to other cell types, promotedproliferation of endothelial cells in vitro, and enhanced angiogenesisand cell migration in the presence of VEGF. These short peptides withregenerative and/or pro-angiogenic properties are useful in promotingangiogenesis, such as in wound healing, burns, tissue repair, fertilitytreatments, myocardial infarction, hypertrophied hearts,revascularization of tissue after disease and trauma (e.g., stroke,ischemic limbs, vascular diseases, bone repair), tissue grafts andtissue engineered constructs. Further, because the peptides and peptidederivatives described herein potentiate the effects of VEGF, they arealso to be considered for their effects on other activities mediated byVEGF. For example, VEGF is a neurotrophic factor that exhibitsneuroprotective properties. The peptides and derivatives describedherein are also useful for promoting nerve growth, neuroprotection,vasodilation, modulation of blood pressure, and treatment of erectiledysfunction.

In one embodiment, provided herein is an isolated peptide fragment of aprominin-1 (prom-1), the peptide having regenerative, pro-angiogenicactivity and/or VEGF-binding activity as measured by, for example, an invitro ELISA-based VEGF-binding assay.

In one embodiment, provided herein is an isolated peptide fragment of aprominin polypeptide, the peptide having regenerative and/orpro-angiogenic activity and also binds to a pro-angiogenic factor.

In one embodiment, provided herein is an isolated peptide fragment of aprominin polypeptide, the peptide having regenerative and/orpro-angiogenic activity and also stimulates endothelial cellproliferation.

In one embodiment, provided herein is an isolated peptide fragment of anextracellular domain of a prominin polypeptide, wherein the peptide hasregenerative and/or pro-angiogenic activity.

In one embodiment, provided herein is isolated peptide fragments of aprominin polypeptide homologue. Homologs of prom-1 are found inzebrafish, Norway rat, house mouse, humans, nematode and fruit fly. In aparticular embodiment, the prominin is human prominin-1 (GenbankAccession No.: NM_(—)006017.1; NP_(—)006008.1; AF027208.1; SEQ ID NO:36).

In some embodiments, the isolated peptide fragments of prom-1 describedherein are derived from the extracellular domain set forth in SEQ. ID.No. 1, 2, or 3.

In some embodiments, the isolated peptide fragments of prom-1 describedherein comprise at least 6 amino acid residues, wherein the peptide doesnot include a full-length prominin polypeptide.

In some embodiments, the isolated peptide fragments of prom-1 describedherein are conservative amino acid substitution variants. To the extentthat a peptide is 12 amino acids or less, such peptides are at least 60%identical to a peptide fragment of prom-1, wherein the peptide variantsbind VEGF and have regenerative and/or pro-angiogenic activity asmeasured by an angiogenesis assay described herein. Further, to theextent that it is known that a given amino acid residue of a prom-1peptide is critical for or implicated in binding, e.g., by mutagenesisor deletion assays, in one embodiment that amino acid residue is notsubstituted. It is specifically contemplated, however, that somesubstitutions at such residues may actually enhance binding. Thus, oneof skill in the art should consider carefully before modifying suchamino acids. Substitutions or modifications to residues of this kindthat maintain or enhance binding to VEGF or the biological properties ofVEGF are also encompassed within the scope of the terms “variant” and“derivative” as used in reference to prom-1 peptides.

In one embodiment, the isolated peptide fragment of a promininpolypeptide, the peptide having regenerative, pro-angiogenic activityand/or VEGF-binding activity as measured by an in vitro ELISA-basedVEGF-binding assay, comprises at least 6 consecutive amino acid residuesfrom e.g., SEQ ID NOs: 1, 2, or 3. While any fragment of prom-1 that hasregenerative and/or pro-angiogenic activity is contemplated, fragmentsof 30 amino acids or less, down to a minimum of 6 amino acids, e.g., 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, or 6 amino acid residues are of particular interest.Preferably, the isolated peptide is selected from the group consistingof LCGNSFSGGQPS (SEQ. ID. No. 4); PNIIPVLDEIKS (SEQ. ID. No. 5);LCGVCGYDRHAT (SEQ. ID. No. 6); ITNNTSSVIIEE (SEQ. ID. No. 7);DRVQRQTTTVVA (SEQ. ID. No. 8); and CSFAYDLEAKANSLPPGNLRN (SEQ. ID. No.9) or a conservative amino acid substitution variant thereof thatsubstantially retains regenerative, pro-angiogenic activity and/orVEGF-binding activity. These peptides dramatically enhance angiogenesisin vivo and enhance cell migration in the presence of VEGF.

Alternatively, or in addition, such peptides or variants or derivativesthereof encompassed by the various embodiments described herein can bindVEGF and exhibit neuroprotective properties.

In one embodiment, the isolated peptide is a VEGF-binding conservativesubstitution variant of a peptide fragment of prominin-1, the peptidehaving regenerative and/or pro-angiogenic activity. The peptide can haveone, two, three or more conservative amino acid substitutions. Thevariant peptide retains a pro-angiogenic activity that is at least 50%of the original non-substituted parent peptide as measured by an invitro ELISA-based VEGF-binding assay.

In another embodiment, the isolated peptide having VEGF-binding andregenerative and/or pro-angiogenic activities is fused with anon-prominin-1 protein, or a heterologous peptide or protein, forming afusion protein comprising a peptide fragment of a prominin-1.

In yet another embodiment, the isolated peptide is a peptide mimetic ofan isolated prom-1 peptide having regenerative and/or pro-angiogenicactivities and binds VEGF.

In yet another embodiment, the isolated peptide having VEGF-binding,regenerative and/or pro-angiogenic activities is conjugated to acompound such as a polymer, e.g., for the purpose of improving serumhalf and pharmacokinetics in vivo. In another embodiment, the isolatedpeptide having VEGF-binding, regenerative and/or pro-angiogenicactivities is fused to another protein or a portion of a protein for thepurpose of improving serum half-life and pharmacokinetics in vivo, forimproved recombinant protein expression and purification, and/orincreased potency of angiogenic activity. The isolated peptide havingVEGF-binding, regenerative and/or pro-angiogenic activities is presentin the context of a fusion protein. In one embodiment, the peptide isPEGylated.

The isolated peptide, variant, fusion protein, peptide mimetic orconjugate described herein enhances VEGF binding to endothelial cells,enhances cell proliferation, enhances angiogenesis in the presence ofpro-angiogenic factors, and enhances cell migration in the presence ofpro-angiogenic factors.

In one embodiment, the isolated peptide, variant, fusion protein,peptide mimetic or conjugate described herein has neuroprotectiveactivity. In another embodiment, the isolated peptide, variant, fusionprotein, peptide mimetic or conjugate described herein prevents ordelays neuronal cell death relative to neuronal cell death occurring inthe absence of the isolated peptide, variant, fusion protein, peptidemimetic or conjugate described herein. In another embodiment, theisolated peptide, variant, fusion protein, peptide mimetic or conjugatedescribed herein promotes nerve regeneration by stimulating neuronalgrowth.

In one embodiment, described herein is a composition comprising apharmaceutically acceptable carrier and an isolated peptide, variant,fusion protein, peptide mimetic or conjugate as described herein.

In one embodiment, a method or use for promoting cell proliferation in atissue in need thereof is provided, the method comprising contacting thetissue with a composition comprising an isolated peptide, variant,peptide mimetic or conjugate thereof.

In one embodiment, one first identifies a tissue in need of e.g., cellproliferation, and then contacts the tissue with a peptide of prom-1, asthat term is used herein.

In one embodiment, a method of promoting angiogenesis in a tissue inneed thereof is provided, the method comprising contacting the tissuewith a composition comprising an isolated peptide, variant, fusionprotein, peptide mimetic or conjugate thereof. The method is applied inthe context of wound healing, burns, tissue repair, bone repair,impaired fertility, myocardial infarction, cardiac hypertrophy, erectiledysfunction, promoting revascularization after disease or trauma, tissuegrafts, or tissue engineered constructs. In addition, the methodsdescribed herein can be administered to potentiate the vasodilatoryeffect of VEGF in the context of disorders associated with reducedvasodilation such as e.g., erectile dysfunction and high blood pressure.

Accordingly, in one embodiment, one first diagnoses the individual ashaving a wound in need of healing, a tissue in need of repair, impairedfertility, cardiac hypertrophy, erectile dysfunction, tissue in need ofrevascularization, tissue in need of grafting or engineered constructs,and then contacts the tissue with a prom-1 peptide as described herein.

Another aspect described herein relates to an isolated prom-1 peptidederived from a prominin polypeptide, the peptide having regenerative,pro-angiogenic and/or VEGF-binding activity.

Also described herein is an isolated prom-1 peptide of a promininpolypeptide, the peptide having regenerative and/or pro-angiogenicactivity, and stimulating endothelial cell proliferation.

Also described herein is an isolated prom-1 peptide derived from anextracellular domain of a prominin polypeptide, the peptide havingregenerative and/or pro-angiogenic activity.

In one embodiment of this aspect and all other aspects described herein,the prominin-1 polypeptide is human prominin-1.

In another embodiment of this aspect and all other aspects describedherein, the extracellular domain is selected from SEQ. ID. No. 1, 2, and3.

In another embodiment of this aspect and all other aspects describedherein, the peptide comprises at least 6 consecutive amino acid residuesof prom-1, e.g., SEQ ID NO: 36, wherein the peptide does not include afull-length prominin polypeptide.

Another aspect described herein relates to an isolated peptide that is aconservative amino acid substitution variant of a peptide as describedabove.

In one embodiment, the peptide of any of the aspects described above isat least 90% identical to a prom-1 peptide, wherein the peptide bindsVEGF and has regenerative and/or pro-angiogenic activity. In otherembodiments, the peptide of any of the aspects described above is atleast 80%, at least 70%, at least 60%, or at least 50% identical to aprom-1 peptide, wherein the peptide binds VEGF and has regenerativeand/or pro-angiogenic activity.

Another aspect described herein relates to an isolated peptide fragmentof a prominin polypeptide, the peptide having regenerative,pro-angiogenic activity and/or VEGF-binding activity, wherein thepeptide consists or consists essentially of a peptide selected from thegroup consisting of: LCGNSFSGGQPS (SEQ. ID. No. 4); PNIIPVLDEIKS (SEQ.ID. No. 5); LCGVCGYDRHAT (SEQ. ID. No. 6); ITNNTSSVIIEE (SEQ. ID. No.7); DRVQRQTTTVVA (SEQ. ID. No. 8); and CSFAYDLEAKANSLPPGNLRN (SEQ. ID.No.9), or a conservative amino acid substitution variant thereof thatsubstantially retains regenerative, pro-angiogenic activity and/orVEGF-binding activity.

In one embodiment, the isolated peptide of any one of the aspectsdescribed herein above enhances VEGF binding to endothelial cells. Thus,methods and uses of the peptide fragments for enhancing VEGF binding toendothelial cells are provided.

In another embodiment, the isolated peptide of any one of the aspectsdescribed herein enhances cell proliferation. Thus, methods and uses ofthe peptide fragments for enhancing cell proliferation are provided.

In another embodiment, the isolated peptide in any of theabove-described aspects enhances proliferation of endothelial cells.Thus, methods and uses of the peptide fragments for enhancingendothelial cell proliferation are provided.

In another embodiment, the isolated peptide in any of theabove-described aspects enhances angiogenesis in the presence of apro-angiogenic factor. Thus, methods and uses of the peptide fragmentsfor promoting angiogenesis are provided.

In another embodiment, the isolated peptide in any of theabove-described aspects enhances cell migration in the presence of apro-angiogenic factor. Thus, methods and uses of the peptide fragmentsfor enhancing cell migration in the presence of a pro-angiogenic factorare provided.

In one embodiment, the isolated prom-1 peptide is a cyclic peptide.

In one embodiment, the cyclic peptide comprises a formula ofCX(DRVQBQTTTVVA)ZC or ACX(DRVQBQTTTVVA)ZC, wherein X or Z are eachindependently 0-20 amino acids, and wherein B is any one of thenaturally occurring amino acid or a derivative thereof. In oneembodiment, the glutamine (Q) at position 6 of the core (DRVQBQTTTVVA)of the cyclic peptide is substituted with any known amino acid otherthan glutamine.

In one embodiment, B is a more hydrophobic amino acid residue thanarginine which has a hydrophobic index of (−4.5) according to Kyte andDoolittle. In another embodiment, the glutamine (Q) at position 6 of thecore (DRVQBQTTTVVA) of the cyclic peptide is substituted with a morehydrophobic amino acid residue than glutamine which has a hydrophobicindex of (−3.5) according to Kyte and Doolittle.

In one embodiment, the cyclic peptide is selected from the groupconsisting of:

ACGG(DRVQRQTTTVVA)GGC, (SEQ ID NO: 15) ACGG(DRVQRQTTTVVA)GGGGGGC,(SEQ ID NO: 16) and CGGGGGG(DRVQRQTTTVVA)GGCA. (SEQ ID NO: 17)

In another embodiment, the isolated peptide in any of theabove-described aspects is conjugated to a polymer.

In another embodiment, a fusion protein comprising a peptide of any ofthe above-described aspects fused to a heterologous peptide orpolypeptide is provided, wherein the fusion protein is not a full-lengthprominin polypeptide.

Another embodiment relates to a composition comprising apharmaceutically acceptable carrier and a peptide or fusion protein ofany one of the above-described aspects.

Also described herein are methods and uses for promoting cellproliferation in a tissue in need thereof, the methods comprisingcontacting the tissue with a composition as described above.

Another aspect described herein relates to a method or use for promotingangiogenesis in a tissue in need thereof, the method comprisingcontacting the tissue with a composition as described above.

In one embodiment of the above-noted aspects, the method is applied inthe context of wound healing, burns, neuronal growth, protection orrepair, tissue repair, bone repair, fertility promotion, myocardialinfarction, cardiac hypertrophy, treatment of erectile dysfunction,modulation of blood pressure, revascularization after disease or trauma,tissue grafts, or tissue engineered constructs.

Also described herein is a method of promoting wound healing, the methodcomprising contacting a wound, or tissue surrounding the wound, with anisolated prom-1 peptide or fusion protein of any one of theabove-described aspects, whereby wound healing is enhanced relative towound healing in the absence of the peptide or fusion protein.

Another aspect described herein relates to a method of promotingneuroprotection, the method comprising contacting a neuronal cell withan isolated prom-1 peptide of a prominin polypeptide or fusion proteinof any one of the above-described aspects, wherein the peptide bindsVEGF, and wherein the contacting promotes neuroprotection of theneuronal cell.

In one embodiment of this aspect and all other aspects described herein,the prom-1 peptide derived from a prominin polypeptide comprisessequence found in an extracellular domain of the prominin polypeptide.

In another embodiment of this aspect and all other aspects describedherein, the prominin polypeptide is human prominin-1.

In another embodiment of this aspect and all other aspects describedherein, the extracellular domain is one of SEQ. ID. No. 1, 2, or 3.

In another embodiment of this aspect and all other aspects describedherein, the prom-1 peptide comprises at least 6 consecutive amino acidresidues SEQ ID NOs: 1, 2, or 3, and wherein the peptide does notinclude a full-length prominin polypeptide.

In another embodiment of this aspect and all other aspects describedherein, the prom-1 peptide comprises a conservative amino acidsubstitution relative to the corresponding wild-type human prominin-1polypeptide sequence.

In another embodiment of this aspect and all other aspects describedherein, the prom-1 peptide is a conservative amino acid substitutionvariant of a peptide of SEQ ID NO: 8.

In another embodiment of this aspect and all other aspects describedherein, the prom-1 peptide comprises or consists essentially of a cyclicpeptide.

In another embodiment of this aspect and all other aspects describedherein, the prom-1 peptide comprises or consists essentially of aheterologous fusion polypeptide.

In another embodiment of this aspect and all other aspects describedherein, the prom-1 peptide is conjugated to a polymer. In oneembodiment, the peptide is PEGylated.

In another embodiment of this aspect and all other aspects describedherein, the prom-1 peptide consists essentially of a peptide of SEQ IDNO: 8.

In another embodiment of this aspect and all other aspects describedherein, the prom-1 peptide consists of SEQ ID NO: 8.

In another embodiment of this aspect and all other aspects describedherein, the contacting step comprises administering a compositioncomprising a prom-1 peptide and a pharmaceutically acceptable carrier toan individual in need thereof, e.g., for neuroprotection. In theembodiment of the method of neuroprotection, one first diagnoses anindividual in need of neuroprotection or neuronal growth, and thencontacts the tissue with a prom-1 peptide as described herein.

In another embodiment of this aspect and all other aspects describedherein, the contacting step prevents or delays neuronal cell deathrelative to neuronal cell death occurring in the absence of thecontacting.

DEFINITIONS

As used herein, the term “regenerative activity” refers to the capacityto stimulate or mediate the restoration of functional tissue followinginsult, disease or disorder that destroys or damages tissue. In oneembodiment, “regenerative activity” refers to lessening, preventingand/or mitigating tissue degeneration resulting from a degenerativedisease, e.g., amyotrophic lateral sclerosis (ALS), multiple sclerosis(MS), Alzheimer's disease, and Parkinson's disease. In one embodiment,“regenerative activity” comprises pro-angiogenic activity. In anotherembodiment, “regenerative activity” comprises neuroprotection and/orstimulation of neuronal growth activity. Assays for “regenerativeactivity” include but are not limited to angiogenesis assays, woundhealing assay, bone repair assay, neuronal growth assay, and use ofmouse models of various diseases (e.g., ALS, MS, Alzheimer's disease,and Parkinson's disease).

As used herein, in one embodiment, the term “pro-angiogenic activity”refers to the stimulation or enhancement of angiogenesis and/orendothelial cell proliferation.

As used herein, the term “prom-1 peptide” refers to a peptide derivedfrom a prominin-1. Preferably, the prom-1 peptide is derived from humanprominin-1 (Genbank Accession No.: NM_(—)006017.1; NP_(—)006008.1;AF027208.1; SEQ ID NO: 36). In one aspect of the invention, the prom-1peptide is not a variant or derivative, but rather consists essentiallyof a peptide of prominin-1 that can potentiate one or more of thebiological effects of an endogenous growth factor. The term “prom-1peptide” encompasses peptides that have regenerative, pro-angiogenic,vasoregulator properties, and/or neurotrophic activity.

As used herein, the term “variant” refers to a peptide fragment fromprom-1 that has one or more conservative amino acid substitutions fromthe sequences of SEQ. ID. NO.4-9. Conservative amino acid substitutionsare well known to one skilled in the art. For example, the amino acidserine can be substituted for threonine and the amino acid aspartate maybe substituted for glutamate. “Conservative amino acid substitutionvariant,” “variant” and “prom-1 peptide variant” are usedinterchangeably herein.

In one aspect, the term “variant” refers to a VEGF-binding peptidefragment from prom-1 that has one or more conservative amino acidsubstitutions from the sequences of SEQ. ID. NO.4-9 but substantiallyretains one or more of the VEGF-binding, regenerative, pro-angiogenic,pro-cell proliferation, pro-cell migration, and/or neuroprotectiveactivities of the original peptide as the specific case may be. By“substantially retain” means the activity of the variant is at least 50%compared to the activity of the original peptide in a similar assay,under similar conditions; preferably the activity is at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, atleast 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least100-fold or higher activity compared to the original peptide. TheVEGF-binding, regenerative, pro-angiogenic, pro-cell proliferation,pro-cell migration, and/or neuroprotective activities of the variantpeptide are determined by methods well known in the art and by themethods described herein. Conservative amino acid substitutions are wellknown to one skilled in the art. For example, the amino acid serine canbe substituted for threonine and the amino acid aspartate may besubstituted for glutamate.

As used herein, a “derivative” of a peptide is a form of a given peptidethat is chemically modified relative to the reference peptide, themodification including, but not limited to, oligomerization orpolymerization, modifications of amino acid residues or peptidebackbone, cross-linking, cyclization, conjugation, fusion to additionalheterologous amino acid sequences, or other modifications thatsubstantially alter the stability, solubility, or other properties ofthe peptide while substantially retaining VEGF binding activity.

As used herein, the term “conservative amino acid substitution” is onein which the amino acid residue is replaced with an amino acid residuehaving a side chain with a similar charge. Families of amino acidresidues having side chains with similar charges have been defined inthe art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Specific examples of conservativeamino acid substitutions are described herein below.

As used herein, the term “peptide mimetic” or “peptidomimetic” refers toa peptide mimetic of a peptide fragment from prom-1 that biologicallymimics the peptide's functions, such as VEGF-binding, regenerative,pro-angiogenic, pro-cell proliferation, pro-cell migration, and/orneuroprotective activities of a prominin-1 peptide as described herein.By “biologically mimics” is meant that a peptidomimetic derivative of apeptide as described herein has at least 50% of the regenerative,pro-angiogenic, pro-proliferative, pro-cell migration, pro-wound healingand/or neuroprotective activity of the peptide itself. In oneembodiment, “biologically mimics” is meant that a peptidomimeticderivative of a peptide as described herein has at least 50% of the VEGFbinding activity and/or at least 50% of the regenerative,pro-angiogenic, pro-proliferative, pro-cell migration, and/orneuroprotective activity of the peptide itself.

As used herein, the term “amino acid” of a peptide refers to naturallyoccurring and synthetic amino acids, as well as amino acid analogs andamino acid mimetics that function in a manner similar to the naturallyoccurring amino acids. Naturally occurring amino acids are those encodedby the genetic code, as well as those amino acids that are latermodified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.Amino acid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acids can be referred toherein by either their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, can be referred to by their commonlyaccepted single-letter codes. In one embodiment, the amino acids in apeptide described herein are naturally occurring amino acids.

By “conjugated” is meant the covalent linkage of at least two molecules.As described herein, an isolated peptide of prom-1 is conjugated to apharmaceutically acceptable polymer to increase its serum half-life.

The term “fragment” refers to any peptide or polypeptide having an aminoacid residue sequence shorter than that of a full-length polypeptidewhose amino acid residue sequence is described herein. An isolatedpeptide of prom-1 is shortened or truncated compared to its parentfull-length prom-1. The polypeptide can have N-terminus or C-terminustruncations and/or also internal deletions. Examples of prom-1 fragmentsinclude fragments consisting of amino acids 13-50, 57-79, and 245-260.

As used herein, the term “homologous proteins” or “homologs” refers toproteins that look similar by way of amino acid sequences and can workin similar ways in different species of organism. For example, human,rabbit, rat, mouse, horse, cow, pig and chicken express transferrins andthese transferrins from the various organisms all have the same functionof transporting iron. The polypeptides of these transferrins areapproximately of the same molecular size and structure, have the samenumber of domains (one N- and one C-terminal domain), again each domainof approximately the same size, and the same number, type and positionof protein secondary folds such as beta-sheets and alpha helices. Whenthe sequences are aligned, homologous proteins have exactly the sameamino acid residues at certain amino acid positions in the polypeptide(i.e., highly conserved regions) and also similar amino acid residues atother amino acid positions in the polypeptide.

As used herein, “heterologous expression” refers to protein expressionin an organism or tissue or cell type that is different from that of thetransgene or coding nucleic acid being expressed into protein in nature.For example, the coding nucleic acid is derived from human, but thecoding nucleic acid is used to express the coded protein is a non-humanorganism (e.g., yeast or hamster) or non-human cells.

As used herein, a “heterologous protein or peptide” refers to a proteinor peptide that is not naturally expressed in an organism or cell. A“heterologous protein” can be expressed when the coding nucleic acidthat codes for it is introduced into the organism that does notnaturally express the “heterologous protein”.

Sequence identity is typically measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various substitutions, deletions,substitutions, and other modifications.

The term “isolated” means the protein is removed from its naturalsurroundings. However, some of the components found with it may continueto be with an “isolated” protein. Thus, an “isolated protein” is not asit appears in nature but may be substantially less than 100% pureprotein.

The term “vector” as used herein, refers to a nucleic acid constructdesigned for delivery to a host cell or transfer between different hostcells. As used herein, a vector can be viral or non-viral. In oneembodiment, the vector permits the expression of a sequence encodedwithin the vector.

As used herein, a “retroviral vector” refers to an expression vectorthat comprises a nucleotide sequence that encodes a transgene and thatfurther comprises nucleotide sequences necessary for packaging of thevector. Preferably, the retroviral transfer vector also comprises thenecessary sequences for expressing the transgene in host cells.

As used herein, the term “pro-angiogenic factors” refers to factors thatdirectly or indirectly promote new blood vessel formation (e.g.,neovascularization).

As used herein, the term “enhances VEGF binding to endothelial cells”refers to an increase in VEGF binding to endothelial cells of at least10% (as assessed by measuring binding of e.g., ¹²⁵I-VEGF to endothelialcells as described herein in the Examples section) in the presence ofboth VEGF and a prom-1 peptide, compared to the amount of VEGF bindingto endothelial cells when VEGF is administered alone. Preferably, theprom-1 peptide enhances VEGF binding to endothelial cells by at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99%, at least1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least100-fold, at least 1000-fold or more compared to VEGF binding when VEGFis administered in the absence of a prom-1 peptide.

As used herein, the terms “increasing angiogenesis”, “promotingangiogenesis” or “enhancing angiogenesis” refer to an increase in atleast one measurable marker of angiogenesis by at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99%, at least1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least100-fold, at least 1000-fold or more, in the presence of a prom-1peptide relative to that marker in the absence of such agent. The termsrecited above also encompass co-treatment of a cell or subject with VEGFand a prom-1 peptide, wherein an increase as described above isdetermined by comparing the angiogenic marker to the effect of VEGFalone on the same marker. To date, six human VEGF mRNA species, encodingVEGF isoforms of 121, 145, 165, 183, 189 and 206 amino acids, areproduced by alternative splicing of the VEGF mRNA.

The determination of VEGF binding to endothelial cells can be performedwith any VEGF isoform that promotes angiogenesis, for example, at leastisoform VEGF₁₆₅, VEGF₁₂₁, and VEGF₁₈₉. In one embodiment, radiolabelledVEGF₁₆₅ is used to determine VEGF binding to endothelial cells.

Endothelial cell migration can be assessed, for example, by measuringthe migration of cells through a porous membrane using a commerciallyavailable kit such as BD BioCoat Angiogenesis System or through a Boydenchamber apparatus. Thus, as used herein, the term “enhances cellmigration” refers, at a minimum, to an increase in the migration ofendothelial cells through a porous membrane of at least 10% in thepresence of a prom-1 peptide; preferably the increase is at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 99%, at least 1-fold, atleast 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, atleast 1000-fold or more in the presence of a prom-1 peptide, as thatterm is used herein.

Endothelial cell growth can be determined, for example, by measuringcell proliferation using an MTS assay commercially available from avariety of companies including RnD Systems, and Promega, among others.Thus, as used herein, the term “enhances cell proliferation” refers toan increase in the number of endothelial cells of at least 10% in thepresence of a prom-1 peptide (as assessed using e.g., an MTS assay);preferably the increase is at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, at least 1-fold, at least 2-fold, at least5-fold, at least 10-fold, at least 100-fold, at least 1000-fold or morein the presence of a prom-1 peptide, as that term is used herein.

The term “wound” as used herein refers broadly to injuries to an organor tissue of an organism that typically involves division of tissue orrupture of a membrane (e.g., skin), due to external violence, amechanical agency, or infectious disease. The term “wound” encompassesinjuries including, but not limited to, lacerations, abrasions,avulsions, cuts, velocity wounds (e.g., gunshot wounds), penetrationwounds, puncture wounds, contusions, hematomas, tearing wounds, bonefractures and/or crushing injuries. In one aspect, the term “wound”refers to an injury to the skin and subcutaneous tissue initiated in anyone of a variety of ways (e.g., pressure sores from extended bed rest,wounds induced by trauma, cuts, ulcers, burns and the like) and withvarying characteristics. Skin wounds are typically classified into oneof four grades depending on the depth of the wound: (i) Grade I: woundslimited to the epithelium; (ii) Grade II: wounds extending into thedermis; (iii) Grade III: wounds extending into the subcutaneous tissue;and (iv) Grade IV (or full-thickness wounds): wounds wherein bones areexposed (e.g., a bony pressure point such as the greater trochanter orthe sacrum).

As used herein, the term “wound healing” refers to a process by whichthe body of a wounded organism initiates repair of a tissue at the woundsite (e.g., skin). In one embodiment, wound healing also includes thehealing of burn wounds. The wound healing process requires, in part,angiogenesis and revascularization of the wounded tissue. Wound healingcan be measured by assessing such parameters as contraction, area of thewound, percent closure, percent closure rate, and/or infiltration ofblood vessels as known to those of skill in the art or as describedherein in the section entitled “Wound healing assays”.

As used herein, the term “treat” or treatment” refers to reducing oralleviating at least one adverse effect or symptom associated withmedical conditions that are associated with excessive, unwanted, and/oraberrant angiogenesis, or diseases or disorders related to degeneration.In one embodiment, “treat” or treatment” refers to reducing oralleviating at least one adverse effect or symptom associated withmedical conditions that are associated with wounds, burns, neuronaldamage, neuron degeneration, cell degeneration, tissue damage, bonedamage, infertility, cardiac hypertrophy, cardiomyopathy, erectiledysfunction and hypertension. In one embodiment, “treat” or treatment”refers to increased collateral artery growth, revascularization afterdisease or trauma and tissue grafts in tissues or subjects in needthereof.

As used herein, the term “promotes neuroprotection” refers to conditionsunder which neuronal cell death (necrotic, apoptotic or otherwise) isprevented or decreased, e.g., by at least 20%, and preferably at least30%, at least 40%, at least 50%, at least 60%, at least 75%, at least90%, at least 95% or more, up to and including, 100% protection in thepresence of an agent such as a peptide disclosed herein, relative to theabsence of that agent. In one aspect, the term refers to conditionsunder which neuronal cell growth, axonal elongation, neuronalproliferation or functional organization is increased by at least 20%,and preferably at least 30%, at least 40%, at least 50%, at least 60%,at least 75%, at least 90%, at least 95% or more, up to and including,for example, at least 1×, at least 2×, at least 3×, at least 5×, atleast 10×, at least 20× or more in the presence of an agent, relative tothe absence of such agent. Effects of neuroprotection can be assessed byany assay known in the art, e.g., neural cell death, neural outgrowthetc. known in the art and/or as described herein.

As used herein, the term “stimulates neuronal growth” refers toconditions under which neuronal cell growth (e.g., axonal growth,trophism) is increased e.g., by at least 20%, and preferably at least30%, at least 40%, at least 50%, at least 60%, at least 75%, at least90%, at least 95% or more, up to and including, for example, at least1×, at least 2×, at least 3×, at least 5×, at least 10×, or at least 20×or more in the presence of an agent, such as a peptide composition setforth herein, relative to the absence of that agent.

As used herein, the term “contacting neurons” refers to any mode ofpeptide delivery or “administration” either to cells, or to wholeorganisms in which the peptide is capable of exhibiting itspharmacological effect in neurons. “Contacting neurons” is intended toinclude both in vivo and in vitro methods of bringing an agent asdescribed herein into proximity with a neuron, particularly in a neuronin need of protection or stimulation, e.g., in neurodegenerative diseasesuch has Parkinson's and ALS. Suitable modes of administration can bedetermined by those skilled in the art, and such modes of administrationmay vary between peptides. For example, when axonal growth of CNSneurons is stimulated ex vivo, peptides can be administered, forexample, by transfection, lipofection, electroporation, viral vectorinfection, or by addition to growth medium. An in vivo means ofcontacting neurons with an agent that stimulate growth of neuronsinclude, but is not limited to, for example, the assay that is describedin Yin et al, 2003, J. Neurosci. 23:2284, which is incorporated byreference in its entirety.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carrierof chemicals and compounds commonly used in the pharmaceutical industry.The term “pharmaceutically acceptable carrier” excludes tissue culturemedium.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in cell biology and molecular biology can be found in “The MerckManual of Diagnosis and Therapy”, 18th Edition, published by MerckResearch Laboratories, 2006 (ISBN 0-911910-18-2); Robert S. Porter etal. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);The ELISA guidebook (Methods in molecular biology 149) by Crowther J. R.(2000); Fundamentals of RIA and Other Ligand Assays by Jeffrey Travis,1979, Scientific Newsletters; Immunology by Werner Luttmann, publishedby Elsevier, 2006. Definitions of common terms in molecular biology arealso be found in Benjamin Lewin, Genes IX, published by Jones & BartlettPublishing, 2007 (ISBN-13: 9780763740634); Kendrew et al. (eds.),Molecular Biology and Biotechnology: a Comprehensive Desk Reference,published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and CurrentProtocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al.,eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Methods in Enzymology,Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon,G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13:978-0121821906); U.S. Pat. Nos. 4,965,343, and 5,849,954; Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al.,Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary protein sequence alignment of some homologousmembers of the prominin family. FIG. 1 discloses SEQ ID NOS 20-26,respectively, in order of appearance.

FIG. 2A shows prominin-1 fragments bind VEGF. Black dots that are abovethe background were chosen as candidates for VEGF binding peptides.

FIG. 2B shows peptide sequences derived from the extracellular domainsof Prominin-1 that bind VEGF. FIG. 2B discloses SEQ ID NOS 4-9,respectively, in order of appearance.

FIG. 3 shows that peptide #237 increased VEGF binding to endothelialcells and melanoma cells. 10000 cells were incubated in binding buffercontaining 20 mM Hepes, 0.1% BSA and I125-VEGF (12 ng/ml) for 3 h onice.

FIG. 3A shows that peptide #237 increased VEGF binding to endothelialcells.

FIG. 3B shows that peptide #237 increased VEGF binding to melanomacells.

FIG. 4: Enhanced VEGF binding is abolished when #237 fragments, #237Aand #237C were used. Prom-1 peptide #237B partially increases the VEGFbinding as compared to the control.

FIG. 5A shows that prominin-1 extracellular fragments increase humanumbilical vein endothelial cell (HUVEC) proliferation.

FIG. 5B shows that prominin-1 extracellular fragments increase humanB16-F10 melanoma cell proliferation.

FIG. 6 shows the effect of Hydron pellets containing VEGF (160ng/pellet) or VEGF+#237 (1.3 ug/pellet) implanted into mouse corneas.Vigorous vessel in-growth from the limbus at 5 days was recorded as apositive response.

FIG. 7 shows that the Prom-1 peptide #237 dramatically increasesendothelial cell migration. Shown are data from the FACS analyses ofmatrigel liberated cells from two groups of treated mice.

FIG. 8A shows the wound healing on a nude mouse ear wound (2.25 mmcircular wound size) with plain MATRIGEL after 5 days. MATRIGEL solutionhad minimal effect on neovascularization.

FIG. 8B shows the wound healing on a nude mouse ear wound (2.25 mmcircular wound size) with MATRIGEL containing the Prom-1 peptide #237(180 μg) after 5 days. The Prom-1 peptide #237 matrigel solutionsignificantly increased the neovascularization around the ear wound site(×4).

FIG. 9 is a bar graph showing the results of experiments indicating thatthe effect of peptides derived from peptide #237 on VEGF binding toendothelial cells is sequence dependent. FIG. 9 discloses SEQ ID NOS 8,8, 8, 8 and 27-35, respectively, in order of appearance.

FIGS. 10A and 10B show that the peptide #237 promotes wound healingafter 14 days in a mouse ear punch model. FIG. 10B is a bar graphshowing the results of the mouse ear punch model experiment.

FIGS. 11A and 11B show that peptide #237 promotes neurite outgrowth inprimary cortical neuronal cells. FIG. 11B is a set of micrographsshowing the results of such experiments.

FIG. 12 shows the laser Doppler images of ischemic mouse tissue treatedwith peptide #237 (SEQ. ID. NO: 8). Animals were ligated in the rightlimb but the ischemic limb is on the left side of the laser Dopplerimages. Black areas indicate no blood flow. White areas indicate bloodflow. Dark shades within the white area at the distal end of limbsindicate high blood flow. SP=scrambled peptide control.

FIG. 13 is a graph showing the blood flow of the ischemic hind limb asthe ratio between the perfusion of the ischemic limb and the uninjuredlimb.

FIG. 14 shows that peptide #237 improves bone repair in a calvariacritical size defect experiment.

FIGS. 15A and 15B are representative histograms showing the effects ofvarious alanine substitutions in the #237 peptides on endothelial cellsbinding to VEGF. Each of the 12 amino acid residues of the original #237peptide was singly replaced by alanine. (Note that the amino acid atposition 12 of original #237 peptide was replaced by glycine in Ala-12).

FIG. 15C discloses twelve alanine substitution #237 peptides, SEQ. ID.NOS. 39-50 respectively, in order of appearance.

DETAILED DESCRIPTION OF THE INVENTION

The methods and compositions described herein are based, in part, on thediscoveries that short peptides derived from the extracellular portionof a penta span transmembrane glycoprotein have distinct effects onangiogenesis. The inventors found that short peptides of Prominin-1(prom-1) bind VEGF (see table 1), an endogenous pro-angiogenesis factorthat is important for normal growth and development but also duringunwanted and aberrant vascularization such as in cancer and diabeticretinopathy. Some of the peptides promoted VEGF binding to other celltypes, promoted proliferation of endothelial cells in vitro, andenhanced angiogenesis and cell migration in the presence of VEGF. Thesepeptides with regenerative and/or pro-angiogenic properties are usefulin promoting angiogenesis such as in wound healing and tissue repair.

The peptides described herein were identified based on their ability tobind VEGF, such as VEGF₁₆₅, VEGF₁₂₁, and VEGF₁₄₅. The peptidespotentiate the activity of VEGF in processes including angiogenesis,cell migration, vasodilation, and cell proliferation. Given theseeffects on the various activities of VEGF, it is considered that thepeptides described herein, and variants and derivatives that bind VEGFcan influence other effect of VEGF, including, for example, neurotrophicand neuroprotective effect, cell migration, cell proliferation, andangiogenesis. In one embodiment, the peptides described herein areadministered to potentiate the vasodilatory effect of VEGF for thetreatment or regulation of high blood pressure and/or erectiledysfunction.

The human prominin-1 (aka AC133, CD133, MSTP061, PROML1, RP41, prominin1, hProminin, prominin (mouse)-like 1, hematopoietic stem cell antigen;Genbank Accession No.: NM_(—)006017.1; NP_(—)006008.1; AF027208.1) is apenta span transmembrane glycoprotein (5-TMD) expressed in stem cells,primarily on the apical membrane of epithelial cells, and is a marker ofhematopoietic stem cells. It belongs to a molecular family of5-transmembrane domain (TMD) proteins, pfam prominin. This “family”includes members from several different species including human, mouse,rat, fly, zebrafish and nematode worms. The 5-TMD structure includes anextracellular N-terminus, two short intracellular loops, two largeextracellular loops and an intracellular C-terminus. Prom-1 wasinitially shown to be expressed on primitive hematopoietic stem andprogenitor cells and on retinoblastoma cells. However, prom-1 has sincebeen shown to be expressed on hemangioblasts, and neural stem cells aswell as on developing epithelia. The prom-1 positive fractions of humanbone marrow, cord blood and peripheral blood efficiently engraft inxenotransplantation models, and contain the majority of thegranulocyte/macrophage precursors, NOD/SCID repopulating cells and CD34+dendritic cell precursors. Phenotypically, prom-1 positive cells inblood and marrow are CD34 bright, with CD34 dim CD71 bright cells beingnegative for prom-1 expression. Prom-1 is also found in extracellularmembrane particles in body fluids. No natural ligand has yet beendemonstrated for prom-1, and its specific function in hematopoietictissue is unknown (Corbeil, D., et. al, Blood. 1998, 91:2625-6; MiragliaS, et. al., Blood. 1997, 90:5013-21; Weigmann A, et. al, Proc Natl.Acad. Sci. USA. 1997, 94:12425-30). The exact function of prominin isunknown although in humans, defects in PROM1, the gene coding forprominin, cause retinal degeneration.

Prom-1 expression is also associated with cancer; many leukemias expressprom-1 as well as CD34. It is believed that colon cancer is initiatedand propagated by a small number of undifferentiated tumorigenic prom-1+cells. Prom-1 mRNA expression is increased in cancer patients withmetastatic disease, specifically with bone metastasis. In addition,prom-1 mRNA expression seems to be an independent prognostic factor foroverall survival.

One of the reasons why prom-1 can promote cancer and/or metastasis isthat the intact full length prom-1 enhances the angiogenesis propertiesof VEGF and therefore is pro-angiogenic. This characteristic of prom-1could certainly support the pathological angiogenesis associated withcancer and metastasis. Treatment options that specifically target theprom-1 protein and its expression are currently under study and includeblocking activity with anti-prom-1 antibody and inhibiting expression bysiRNA.

Provided herein are peptides derived from a prominin polypeptide thathas regenerative and/or pro-angiogenic activities.

Accordingly, embodiments of the present invention provide isolatedpeptide fragments derived from prominin-1 (prom-1), the peptides havingregenerative, pro-angiogenic activity and/or VEGF-binding activity asmeasured by an in vitro ELISA-based VEGF-binding assay. Thepro-angiogenic activities include: promoting VEGF binding to other celltypes, the other cell types including, for example, endothelial cells;promoting cell proliferation of endothelial cells in vitro; enhancingangiogenesis in the presence of VEGF as assayed via a corneal micropocket assay; enhancing endothelial cell migration in vivo in thepresence of VEGF; and promoting neovascularization in vivo in thepresence of growth factors as assayed in an ear wound healing assay asdescribed herein.

In one embodiment, an isolated peptide with regenerative and/orpro-angiogenic activity is derived from the full length human prom-1(Genbank Accession No.: NM_(—)006017.1; NP_(—)006008.1; AF027208.1).However, under no circumstances does the term “prom-1 peptide” encompassthe full length peptide. Rather, “prom-1 peptide” refers to a truncatedpeptide of prom-1. In other embodiments, an isolated peptide withregenerative and/or pro-angiogenic activity is from homologous proteinmembers of the prominin protein family (pfam05478:Prominin). Homologousprotein members of the prominin protein family include known andidentified proteins as well as predicted proteins from genomic studies.All members of the prominin family are predicted to contain fivemembrane spanning domains, with an N-terminal domain exposed to theextracellular space followed by four, alternating small cytoplasmic andlarge extracellular loops and a cytoplasmic C-terminal domain. Theseproteins are homologs of the human prom-1. An example of the proteinsequence alignment of some of the prominin family members is shown inFIG. 1. Examples of prom-1 homologs are: Danio rerio (zebrafish)prominin-like 2 protein, swissprot Q90WI3, Genbank Accession No.GI:82177379; Rattus norvegicus (Norway rat) testosterone-regulatedprominin-related protein, swissprot Q8R4B6, Genbank Accession No.GI:81866961; Homo sapiens (human) Prominin-like protein 2, swissprotQ8N271, Genbank Accession No. GI:74728673; Mus musculus (house mouse)Prominin-like protein 1, swissprot 054990, Genbank Accession No.GI:13124464; Caenorhabditis elegans hypothetical protein F08B12.1,swissprot Q19188, Genbank Accession No. GI:74963586; and Drosophilamelanogaster (fruit fly) Prominin-like protein, swissprot P82295,Genbank Accession No. GI:13124468.

In one embodiment, the isolated peptide with regenerative and/orpro-angiogenic activity comprises at least 6 consecutive amino acidresidues. The 6 amino acid residues are consecutive, reflecting what isencoded and expressed in the intact full length prom-1 polypeptide. Inanother embodiment, the isolated peptide with regenerative and/orpro-angiogenic activity comprises no more than 50 amino acids of prom-1.In yet another embodiment, the isolated peptide with pro-angiogenicactivity has at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive amino acidresidues of prom-1. In another embodiment, the isolated peptide has nomore than 30 consecutive amino acid residues. In yet another embodiment,the isolated peptide has no more than 29, 28, 27, 26, 25, 24, 23, 22,21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 consecutiveamino acid residues of prom-1. In another embodiment, the peptide has 50or fewer consecutive amino acids and includes peptides with 49, 48, 47,46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, and 30consecutive amino acids of prom-1.

In one aspect, the isolated peptide with regenerative and/orpro-angiogenic activity consists essentially of or consists of a peptideselected from the group consisting of: LCGNSFSGGQPS (SEQ. ID. No. 4);PNIIPVLDEIKS (SEQ. ID. No. 5); LCGVCGYDRHAT (SEQ. ID. No. 6);ITNNTSSVIIEE (SEQ. ID. No. 7); DRVQRQTTTVVA (SEQ. ID. No. 8); andCSFAYDLEAKANSLPPGNLRN (SEQ. ID. No. 9) or a conservative substitutionvariant thereof.

Encompassed herein is an isolated peptide that is a VEGF-bindingconservative substitution variant of a peptide with regenerative and/orpro-angiogenic activity as measured by an in vitro ELISA-basedVEGF-binding assay as described herein. Conservative amino acid residuesubstitution is well known in the art. Conservative amino acidsubstitutions replace an amino acid with another amino acid of similarchemical structure. Examples of such substitution are glycine foralanine, leucine for valine, serine for threonine, and aspartate forglutamate. For example, SEQ ID NO: 4 can be altered to a peptideVCGNSFSGGQPS, LCANSFSGGQPS, or LCGNSFSAGQPS, or LCGNSFSGAQPS where theresidues that are in bold and italicized are changed. In one embodiment,only one of the amino acids is substituted with a conservativesubstitution. In another embodiment, two substitutions can be made. Inother embodiments, up to four amino acids are substituted. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. Conservative amino acidsubstitutions do not change the overall structure of the peptide nor thetype of amino acid side chains available for forming van der Waals bondswith a binding partner. Conservative amino acid substitution can beachieved during chemical synthesis of the peptide by adding the desiredsubstitute amino acid at the appropriate sequence in the synthesisprocess. Alternatively, molecular biology methods can be used. Thecoding sequence of a peptide described herein can be made by annealingtwo single strand nucleic acids that are complementary to each other.The sequence of the nucleic acid codes for the peptide. For example, asense nucleic acid for the prom-1 peptide LCGNSFSGGQPS (SEQ. ID. No. 4)is 5′ CTGTGCGGCAACAGCTTTAGCGGCGGCCAGCCGAGC 3′(SEQ. ID. No.10) and thecomplementary anti-sense nucleic acid is 5′GCTCGGCTGGCCGCCGCTAAAGCTGTTGCCGCACAG 3′(SEQ. ID. No. 11). After decidingon a substitute amino acid, the triplet codon for that substitute aminoacid is determined and incorporated into the design of two complementarysingle strand nucleic acid sequences, with the codon of the substituteamino acid replacing the codon of amino acid that is being substituted.For example, for a serine to threonine substitution of the serine inposition 5′ of LCGNSFSGGQPS (SEQ. ID. No. 4), the triplet codon AGC forserine is replaced with the triplet codon ACG for threonine in the SEQ.ID. No. 10. Complementary changes in the design of the anti-sense SEQ.ID. No.11 are then made in order to anneal the two strands.Alternatively, site-directed mutagenesis, a known art, can be used forconservative amino acid substitution in the peptides described herein.

In some embodiments, conservative amino acid substitution are not to befound in the amino acid residues that are important for itsregenerative, pro-angiogenic activity, pro-cell proliferation, and/orcell migration activity. In one embodiment, when a conservative aminoacid substitution occurring at position X in a peptide results inreduced regenerative, pro-angiogenic activity, pro-cell proliferation,and/or cell migration activity, it is an indication that the amino acidresidue at position X is important for its regenerative, pro-angiogenicactivity, pro-cell proliferation, and/or cell migration activity. Forexample, in DRVQRQTTTVVA (SEQ. ID. NO:8), substitution of the valine atposition 10 this amino acid with the conservative amino acid alaninedestroyed activity and should not be substituted (See Example 13, FIG.15).

In one embodiment, the isolated peptide and VEGF-binding conservativeamino acid substitution variant thereof with regenerative and/orpro-angiogenic activity described herein enhances VEGF binding toendothelial cells. In another embodiment, the peptide with regenerativeand/or pro-angiogenic activity described herein enhances cellproliferation in endothelial cells. In another embodiment, the peptidewith regenerative and/or pro-angiogenic activity described hereinenhances angiogenesis in the presence of pro-angiogenic factors. In someaspects, the peptide with regenerative and/or pro-angiogenic activitydescribed herein enhances cell migration, such as endothelial cells, inthe presence of pro-angiogenic factors. In another aspect, the peptidewith regenerative and/or pro-angiogenic activity described hereinpromotes neovascularization in vivo in the presence of growth factors.Such growth factors include, but are not limited to, VEGF, EGF, bFGF,NGF, PDGF, IGF-1, and TGF-β.

In one embodiment, the isolated prom-1 peptide is a cyclic peptide.

In one embodiment, the cyclic peptide comprises a formula ofCX(DRVQBQTTTVVA)ZC or ACX(DRVQBQTTTVVA)ZC, wherein X or Z are eachindependently 0-20 amino acids, and wherein B is any one of thenaturally occurring amino acid or a derivative thereof. In oneembodiment, the glutamine (Q) at position 6 of the core (DRVQBQTTTVVA)of the cyclic peptide is substituted with any one of the known 20 aminoacids other that glutamine.

In one embodiment, B is a more hydrophobic amino acid residue thanarginine which has a hydrophobic index of (−4.5) according to Kyte andDoolittle. In another embodiment, the glutamine (Q) at position 6 of thecore (DRVQBQTTTVVA) of the cyclic peptide is substituted with a morehydrophobic amino acid residue than glutamine which has a hydrophobicindex of (−3.5) according to Kyte and Doolittle.

In one embodiment, the cyclic peptide is selected from the groupconsisting of:

ACGG(DRVQRQTTTVVA)GGC, (SEQ ID NO: 15) ACGG(DRVQRQTTTVVA)GGGGGGC,(SEQ ID NO: 16) and CGGGGGG(DRVQRQTTTVVA)GGCA. (SEQ ID NO: 17)

In one embodiment, a fusion polypeptide comprising a prom-1 peptide or aconservative amino acid substitution variant thereof is contemplatedherein. The fusion polypeptide is formed by the fusion of a peptidedescribed herein with another heterologous protein or a portion thereof.The heterologous protein is any protein that is not a member of theprominin family. The fusion gives rise to a prom-1 chimeric polypeptide.Such fusion prom-1 peptides can serve to enhance the serum half life ofthe prom-1 peptide in vivo. Examples include fusion with albumin,transferrin, transthyretin, and Fc of IgG (See G. M. Subramanian, 2007,Nature Biotechnology 25, 1411-141). Other fusions can facilitate proteinexpression, solubility during expression, and purification, e.g.,thioredoxin, glutathione S-transferase, avidin and six histidine tags(SEQ ID NO: 19). In another embodiment, a peptide or a conservativeamino acid substitution variant thereof described herein withregenerative and/or pro-angiogenic activity can be fused with otherpro-angiogenic factors, e.g., VEGF, FGF and IGF to enhance angiogenicpotency.

Peptide Modifications

It is to be understood that modified versions of the peptides describedherein are encompassed in the present invention. Conservativesubstitutions are discussed herein above. Non-conservative substitutionsare encompassed to the extent that that they substantially retain theactivities of those peptides. Modification to a prom-1 peptide can beperformed as described in U.S. published patent application Nos.20080090760 and 20060286636, each of which is incorporated herein byreference in its entirety. The following provides a non-limitingdiscussion of various other peptide modifications encompassed within thescope of the invention.

Encompassed by the peptide described herein are chemical derivatives ofa prom-1 peptide whose amino acid residue sequence is described herein,so long as they substantially retain the activities of those peptides. A“chemical derivative” is a subset of peptide derivatives as describedherein and refers to a subject polypeptide having one or more residueschemically derivatized by reaction of a functional side group. Inaddition to side group derivatizations, a chemical derivative can haveone or more backbone modifications including alpha-amino substitutionssuch as N-methyl, N-ethyl, N-propyl and the like, and alpha-carbonylsubstitutions such as thioester, thioamide, guanidino and the like. Suchderivatized molecules include for example, those molecules in which freeamino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids.Also included as chemical derivatives are those peptides which containone or more non-limiting, non-natural amino acids, examples includethose available for peptide synthesis from commercial suppliers (e.g.,Bachem Catalog, 2004 pp. 1-276). For examples: 4-hydroxyproline may besubstituted for proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; ornithine may be substituted for lysine;β-alanine may be substituted for alanine; norleucine may be substitutedfor leucine; phenylglycine may be substituted for phenylalanine, andL-1,2,3,4-tetrahydronorharman-3-carboxylic acid orH-β-(3-Benzothienyl)-Ala-OH may be substituted for tryptophan.

In certain embodiments, chemical modifications to the peptide include,but are not limited to the inclusion of, alkyl, alkoxy, hydroxyalkyl,alkoxyalkyl, alkoxycarbonyl, alkenyl, alkynyl, cycloalkyl, amino,alkylamino, aminoalkyl, dialkylamino, aminodialkyl, halogen, heteroatom,carbocycle, carbocyclyl, carbocyclo, carbocyclic, aryl, aralkyl,aralkoxy, aryloxyalkyl, heterocycle, heterocyclyl, heterocyclic,heteroaryl, and/or aliphatic groups.

The terms “alkyl”, “alkoxy”, “hydroxyalkyl”, “alkoxyalkyl”, and“alkoxycarbonyl”, used alone or as part of a larger moiety includes bothstraight and branched chains containing one to twelve carbon atoms. Theterms “alkenyl” and “alkynyl” used alone or as part of a larger moietyshall include both straight and branched chains containing two to twelvecarbon atoms. The term “cycloalkyl” used alone or as part of a largermoiety shall include cyclic C₃-C₁₂ hydrocarbons which are completelysaturated or which contain one or more units of unsaturation, but whichare not aromatic. Lower alkyl refers to an alkyl group containing 1-6carbons.

The term “amino” refers to an NH₂ group.

The term “alkylamino” or “aminoalkyl” refers to an amino group whereinone of the hydrogen atoms is replaced by an alkyl group.

The term “dialkylamino” or “aminodialkyl” refers to an amino groupwherein the hydrogen atoms are replaced by alkyl groups, wherein thealkyl group may be the same or different.

The term “halogen” means F, Cl, Br, or I.

The term “heteroatom” means nitrogen, oxygen, or sulfur with a carbonring structure and includes any oxidized form of nitrogen and sulfur,and the quaternized form of any basic nitrogen. Also the term “nitrogen”includes substitutable nitrogen of a heterocyclic ring. As an example,in a saturated or partially unsaturated ring having 0-3 heteroatomsselected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as inN-substituted pyrrolidinyl).

The terms “carbocycle”, “carbocyclyl”, “carbocyclo”, or “carbocyclic” asused herein means an aliphatic ring system having three to fourteenmembers. The terms “carbocycle”, “carbocyclyl”, “carbocyclo”, or“carbocyclic” whether saturated or partially unsaturated, also refers torings that are optionally substituted. The terms “carbocycle”,“carbocyclyl”, “carbocyclo”, or “carbocyclic” also include aliphaticrings that are fused to one or more aromatic or nonaromatic rings, suchas in a decahydronaphthyl or tetrahydronaphthyl, where the radical orpoint of attachment is on the aliphatic ring.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to aromatic ring groupshaving six to fourteen members, such as phenyl, benzyl, phenethyl,1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl. The term “aryl”also refers to rings that are optionally substituted. The term “aryl”may be used interchangeably with the term “aryl ring”. “Aryl” alsoincludes fused polycyclic aromatic ring systems in which an aromaticring is fused to one or more rings. Examples include 1-naphthyl,2-naphthyl, 1-anthracyl and 2-anthracyl. Also included within the scopeof the term “aryl”, as it is used herein, is a group in which anaromatic ring is fused to one or more non-aromatic rings, such as in anindanyl, phenanthridinyl, or tetrahydronaphthyl, where the radical orpoint of attachment is on the aromatic ring.

The term “heterocycle”, “heterocyclyl”, or “heterocyclic” as used hereinincludes non-aromatic ring systems having four to fourteen members,preferably five to ten, in which one or more ring carbons, preferablyone to four, are each replaced by a heteroatom. Examples of heterocyclicrings include 3-1H-benzimidazol-2-one,(1-substituted)-2-oxo-benzimidazol-3-yl, 2-tetrahydro-furanyl,3-tetrahydrofuranyl, 2-tetrahydropyranyl, 3-tetrahydropyranyl,4-tetra-hydropyranyl, [1,3]-dioxalanyl, [1,3]-dithiolanyl,[1,3]-dioxanyl, 2-tetra-hydro-thiophenyl, 3-tetrahydrothiophenyl,2-morpholinyl, 3-morpholinyl, 4-morpholinyl, 2-thiomorpholinyl,3-thiomorpholinyl, 4-thiomorpholinyl, 1-pyrrolidinyl, 2-pyrrolidinyl,3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl, 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 4-thiazolidinyl,diazolonyl, N-substituted diazolonyl, 1-phthalimidinyl, benzoxanyl,benzopyrrolidinyl, benzopiperidinyl, benzoxolanyl, benzothiolanyl, andbenzothianyl. Also included within the scope of the term “heterocyclyl”or “heterocyclic”, as it is used herein, is a group in which anon-aromatic heteroatom-containing ring is fused to one or more aromaticor non-aromatic rings, such as in an indolinyl, chromanyl,phenanthridinyl, or tetrahydroquinolinyl, where the radical or point ofattachment is on the non-aromatic heteroatom-containing ring. The term“heterocycle”, “heterocyclyl”, or “heterocyclic” whether saturated orpartially unsaturated, also refers to rings that are optionallysubstituted.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to heteroaromatic ringgroups having five to fourteen members. Examples of heteroaryl ringsinclude 2-furanyl, 3-furanyl, 3-furazanyl, N-imidazolyl, 2-imidazolyl,4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl,2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 1-pyrazolyl, 2-pyrazolyl,3-pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-pyrimidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,5-tetrazolyl, 2-triazolyl, 5-triazolyl, 2-thienyl, 3-thienyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, indazolyl, isoindolyl, acridinyl, andbenzoisoxazolyl. Also included within the scope of the term“heteroaryl”, as it is used herein, is a group in which a heteroatomicring is fused to one or more aromatic or nonaromatic rings where theradical or point of attachment is on the heteroaromatic ring. Examplesinclude tetrahydroquinolinyl, tetrahydroisoquino-linyl, andpyrido[3,4-d]pyrimidinyl. The term “heteroaryl” also refers to ringsthat are optionally substituted. The term “heteroaryl” may be usedinterchangeably with the term “heteroaryl ring” or the term“heteroaromatic”.

An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) orheteroaryl (including heteroaralkyl and heteroarylalkoxy and the like)group may contain one or more substituents. Examples of suitablesubstituents on any unsaturated carbon atom of an aryl, heteroaryl,aralkyl, or heteroaralkyl group include a halogen, —R0, —OR0, —SR0,1,2-methylene-dioxy, 1,2-ethylenedioxy, protected OH (such as acyloxy),phenyl (Ph), substituted Ph, —O(Ph), substituted —O(Ph), —CH2(Ph),substituted —CH2(Ph), CH2CH2(Ph), substituted —CH2CH2(Ph), —NO2, —CN,—N(R0)2, —NR0C(O)R0, NR0C(O)N(R0)2, NR0CO2R0, —NR0NR0C(O)R0,—NR0NR0C(O)N(R0)2, —NR0NR0C2R0, C(O)C(O)R0, C(O)CH2C(O)R0, —CO2R0,—C(O)R0, —C(O)N(R0)2, —OC(O)N(R0)2, S(O)2R0, —SO2N(R0)2, —S(O)R0,—NR0SO2N(R0)2, —NR0SO2R0, —C(═S)N(R0)2, C(═NH)N(R0)2, (CH2)yNHC(O)R0,and —(CH2)yNHC(O)CH(V—R0)(R0); wherein each R0 is independently selectedfrom hydrogen, a substituted or unsubstituted aliphatic group, anunsubstituted heteroaryl or heterocyclic ring, phenyl (Ph), substitutedPh, O(Ph), substituted —O(Ph), —CH2 (Ph), or substituted —CH2(Ph); y is0-6; and V is a linker group. Examples of substituents on the aliphaticgroup or the phenyl ring of R0 include amino, alkylamino, dialkylamino,aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano,carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, andhaloalkyl.

An aliphatic group or a non-aromatic heterocyclic ring or a fused arylor heteroaryl ring may contain one or more substituents. Examples ofsuitable substituents on any saturated carbon of an aliphatic group orof a non-aromatic heterocyclic ring or a fused aryl or heteroaryl ringinclude those listed above for the unsaturated carbon of an aryl orheteroaryl group and the following: ═O, ═S, ═NNHR*, ═NN(R*)2, ═N—,═NNHC(O)R*, ═NNHCO2(alkyl), ═NNHSO2 (alkyl), or ═NR*, where each R* isindependently selected from hydrogen, an unsubstituted aliphatic group,or a substituted aliphatic group. Examples of substituents on thealiphatic group include amino, alkylamino, dialkylamino, aminocarbonyl,halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano,carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, andhaloalkyl.

Suitable substituents on the nitrogen of a non-aromatic heterocyclicring include R+, —N(R+)2, —C(O)R+, —CO2R+, —C(O)C(O)R+, —C(O)CH2C(O)R+,—SO2R+, —SO2N(R+)2, C(═S)N(R+)2, —C(═NH)—N(R+)2, and —NR+SO2R+; whereineach R+ is independently selected from hydrogen, an aliphatic group, asubstituted aliphatic group, phenyl (Ph), substituted Ph, —O(Ph),substituted —O(Ph), —CH2(Ph), substituted —CH2(Ph), or an unsubstitutedheteroaryl or heterocyclic ring. Examples of substituents on thealiphatic group or the phenyl ring include amino, alkylamino,dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylaminocarbonyloxy, dialkylaminocarbonyloxy,alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy,haloalkoxy, and haloalkyl.

In certain embodiments, the peptide monomers described herein aredimerized or multimerized by covalent attachment to at least one linkermoiety. The linker moiety is preferably, although not necessarily, aC₁₋₁₂ linking moiety optionally terminated with one or two —NH—linkagesand optionally substituted at one or more available carbon atoms with alower alkyl substituent. Preferably the linker comprises —NH—R—NH—wherein R is a lower (C₁₋₆) alkylene substituted with a functionalgroup, such as a carboxyl group or an amino group, that enables bindingto another molecular moiety (e.g., as may be present on the surface of asolid support during peptide synthesis or to a pharmacokinetic-modifyingagent such as PEG). In certain embodiments the linker is a lysineresidue. In certain other embodiments, the linker bridges the C-terminiof two peptide monomers, by simultaneous attachment to the C-terminalamino acid of each monomer. In other embodiments, the linker bridges thepeptides by attaching to the side chains of amino acids not at theC-termini. When the linker attaches to a side chain of an amino acid notat the C-termini of the peptides, the side chain preferably contains anamine, such as those found in lysine, and the linker contains two ormore carboxy groups capable of forming an amide bond with the peptides.

The peptide monomers of the invention may be oligomerized using thebiotin/streptavidin system. Oligomerizat6ion can enhance one or moreactivities of peptides as described herein. Biotinylated analogs ofpeptide monomers may be synthesized by standard techniques known tothose skilled in the art. For example, the peptide monomers may beC-terminally biotinylated. These biotinylated monomers are thenoligomerized by incubation with streptavidin (e.g., at a 4:1 molar ratioat room temperature in phosphate buffered saline (PBS) or HEPES-bufferedRPMI medium (Invitrogen) for 1 hour). In a variation of this process,biotinylated peptide monomers may be oligomerized by incubation with anyone of a number of commercially available anti-biotin antibodies [e.g.,goat anti-biotin IgG from Kirkegaard & Perry Laboratories, Inc.(Washington, D.C.)].

In some aspects, the peptides described herein can be linked physicallyin tandem to form a polymer of prom-1 peptides. The peptides making upsuch a polymer can be spaced apart from each other by a peptide linker.A “peptide linker” is a short (e.g., about 1-40, e.g., 1-20 amino acids)sequence of amino acids that is not part of the prom-1 or variantsequence. A linker peptide is attached on its amino-terminal end to onepolypeptide or polypeptide domain and on its carboxyl-terminal end toanother polypeptide or polypeptide domain. Examples of useful linkerpeptides include, but are not limited to, glycine polymers ((G)n)including glycine-serine and glycine-alanine polymers (e.g., a(Gly₄Ser)n repeat where n=1-8 (SEQ ID NO: 18), preferably, n=3, 4, 5, or6). The prom-1 peptides described herein can also be joined by chemicalbond linkages, such as linkages by disulfide bonds or by chemicalbridges. Molecular biology techniques that are well known to thoseskilled in the art can be used to create a polymer of prom-1 peptides.In one embodiment, combination of a prom-1 peptide and variant peptideis found in the polymer. Peptide sequences of the present invention canalso be linked together using non-peptide cross-linkers (Pierce2003-2004 Applications Handbook and Catalog, Chapter 6) or otherscaffolds such as HPMA, polydextran, polysaccharides, ethylene-glycol,poly-ethylene-glycol, glycerol, sugars, and sugar alcohols (e.g.,sorbitol, mannitol).

In an optional embodiment, polyethylene glycol (PEG) may serve as alinker that dimerizes two peptide monomers: for example, a single PEGmoiety containing two reactive functional groups may be simultaneouslyattached to the N-termini of both peptide chains of a peptide dimer.These peptides are referred to herein as “PEGylated peptides.”

In yet another embodiment, a linker moiety may comprise a moleculecontaining two carboxylic acids and optionally substituted at one ormore available atoms with an additional functional group such as anamine capable of being bound to one or more PEG molecules. Such amolecule can be depicted as:—CO—(CH₂)n-uX—(CH₂)m-CO— where n is aninteger between zero and 10, m is an integer between one and 10, X isselected from O, S, N(CH₂)pNR1, NCO(CH₂)pNR1, and CHNR1, R1 is selectedfrom H, Boc (tert-butyloxycarbonyl), Cbz, and p is an integer between 1and 10. In certain embodiments, one amino group of each of the peptidesforms an amide bond with the linker. In certain other embodiments, theamino group of the peptide bound to the linker is the epsilon amine of alysine residue or the alpha amine of the N-terminal residue, or an aminogroup of an optional spacer molecule. In one embodiment, a linker isused to cyclize peptides. In another embodiment, a spacer can be used inaddition to a linker molecule for separating moieties as desired. Inparticularly preferred embodiments, both n and m are one, X isNCO(CH₂)pNR1, p is two, and R1 is Boc. Optionally, the Boc group can beremoved to liberate a reactive amine group capable of forming a covalentbond with a suitably activated PEG species such as mPEG-SPA-NHS ormPEG-NPC (Nektar Therapeutics, San Carlos Calif.). Optionally, thelinker contains more than one reactive amine capable of beingderivatized with a suitably activated PEG species. Optionally, thelinker contains one or more reactive amines capable of being derivatizedwith a suitably activated pharmacokinetic (PK) modifying agent such as afatty acid, a homing peptide, a transport agent, a cell-penetratingagent, an organ-targeting agent, or a chelating agent.

A peptide monomer, dimer, multimer or oligomer as described herein mayfurther comprise one or more linker and/or spacer moieties. In oneembodiment, the linker moiety is a C₁₋₁₂ linking moiety optionallyterminated with —NH— linkages or carboxyl (—COOH) groups, and optionallysubstituted at one or more available carbon atoms with a lower alkylsubstituent. In one embodiment, the linker is R—COOH wherein R is alower (C1-6) alkyl optionally substituted with a functional group suchas a carboxyl group or an amino group that enables binding to anothermolecular moiety. For example, the linker may be a glycine (G) residue,or an amino hexanoic acid (Ahx) such as 6-amino hexanoic acid. In otherembodiments, the linker is —NH—R—NH— wherein R is a lower (C1-6) alkylsubstituted with a functional group such as a carboxyl group or an aminogroup that enables binding to another molecular moiety. For example, thelinker may be a lysine (K) residue or a lysine amide (K—NH₂, a lysineresidue wherein the carboxyl group has been converted to an amide moiety—CONH₂).

In some embodiments, the linker moiety has the following structure:—NH—(CH₂)_(α)—[O—(CH₂)_(β)]_(γ)—O_(δ)—(CH₂)_(ε)—Y— where α, β, γ, δ, andε are each integers whose values are independently selected. In someembodiments, α, β, and c are each integers whose values areindependently selected between one and about six, δ is zero or one, γ isan integer selected between zero and about ten, except that when γ isgreater than one, β is two, and Y is selected from NH or CO. In someembodiments, α, β, and c are each equal to two, both γ and δ are equalto 1, and Y is NH. In another embodiment, γ and 8 are zero, α and εtogether equal five, and Y is CO.

The peptide monomers, dimers, or multimers of the invention may furthercomprise one or more water soluble polymer moieties. Preferably, thesepolymers are covalently attached to the peptide compounds of theinvention. Preferably, for therapeutic use of the end productpreparation, the polymer is pharmaceutically acceptable. One skilled inthe art will be able to select the desired polymer based on suchconsiderations as whether the polymer-peptide conjugate will be usedtherapeutically, and if so, the desired dosage, circulation time,resistance to proteolysis, and other considerations. The water solublepolymer may be, for example, polyethylene glycol (PEG), copolymers ofethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers),poly(n-vinyl-pyrrolidone)polyethylene glycol, propropylene glycolhomopolymers, polypropylene oxide/ethylene oxide copolymers, andpolyoxyethylated polyols. A preferred water soluble polymer is PEG.

The polymer may be of any molecular weight, and may be branched orunbranched. A preferred PEG for use in the present invention is linear,unbranched PEG having a molecular weight of from about 5 kilodaltons(kDa) to about 60 kDa (the term “about” indicating that in preparationsof PEG, some molecules will weigh more, and some less, than the statedmolecular weight). More preferably, the PEG has a molecular weight offrom about 10 kDa to about 40 kDa, and even more preferably, the PEG hasa molecular weight from 20 to 30 kDa. Other sizes may be used, dependingon the desired therapeutic profile (e.g., duration of sustained releasedesired; effects, if any, on biological activity; ease in handling;degree or lack of antigenicity; and other effects of PEG on atherapeutic peptide known to one skilled in the art).

The number of polymer molecules attached may vary; for example, one,two, three, or more water-soluble polymers may be attached to a peptideof the invention. The multiple attached polymers may be the same ordifferent chemical moieties (e.g., PEGs of different molecular weight).

In certain embodiments, PEG may be attached to at least one terminus(N-terminus or C-terminus) of a peptide monomer or dimer. In otherembodiments, PEG may be attached to a linker moiety of a peptide monomeror dimer. In a preferred embodiment, PEG is attached to the linkermoiety of a peptide dimer. Optionally, the linker contains more than onereactive amine capable of being derivatized with a suitably activatedPEG species.

Methods for stabilizing peptides known in the art may be used with themethods and compositions described herein. For example, using D-aminoacids, using reduced amide bonds for the peptide backbone, and usingnon-peptide bonds to link the side chains, including, but not limitedto, pyrrolinone and sugar mimetics can each provide stabilization. Thedesign and synthesis of sugar scaffold peptide mimetics are described byHirschmann et al. (J. Med. Chem., 1996, 36, 2441-2448, which isincorporated herein by reference in its entirety). Further,pyrrolinone-based peptide mimetics present the peptide pharmacophore ona stable background that has improved bioavailability characteristics(see, for example, Smith et al., J. Am. Chem. Soc. 2000, 122,11037-11038), which is incorporated herein by reference in its entirety.

Encompassed herein are conjugates of a peptide described herein or of aconservative amino acid substitution variant or derivative thereof.These peptides can be conjugated to other polymers in addition topolyethylene glycol (PEG). The polymer may or may not have its ownbiological activity. Further examples of polymer conjugation include butare not limited to polymers such as polyvinyl pyrrolidone, polyvinylalcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polyoxyethylatedpolyol, heparin, heparin fragments, polysaccharides, cellulose andcellulose derivatives, including methylcellulose and carboxymethylcellulose, starch and starch derivatives, polyalkylene glycol andderivatives thereof, copolymers of polyalkylene glycols and derivativesthereof, polyvinyl ethyl ethers, andα,β-Poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixturesthereof. Conjugation to a polymer can improve serum half-life, amongother effects. A variety of chelating agents can be use to conjugate thepeptides described herein. These chelating agents include but are notlimited to ethylenediaminetetraacetic acid (EDTA),diethylenetriaminopentaacetic acid (DTPA),ethyleneglycol-0,0′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA),N,N′-bis(hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED),triethylenetetraminehexaacetic acid (TTHA),1,4,7,10-tetra-azacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA),1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetraacetic acid (TITRA),1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA),and 1,4,8,11-tetraazacyclotetradecane (TETRA). Methods of conjugationare well known in the art, for example, P. E. Thorpe, et. al, 1978,Nature 271, 752-755; Harokopakis E., et. al., 1995, Journal ofImmunological Methods, 185:31-42; S. F. Atkinson, et al., 2001, J. Biol.Chem., 276:27930-27935; and U.S. Pat. Nos. 5,601,825, 5,180,816,6,423,685, 6,706,252, 6,884,780, and 7,022,673, which are herebyincorporated by reference in their entirety.

In one embodiment, the peptides, fusion proteins or conjugates of prom-1peptides include modifications within the sequence, such as,modification by terminal-NH₂ acylation, e.g., acetylation, orthioglycolic acid amidation, by terminal-carboxylamidation, e.g., withammonia, methylamine, and the like terminal modifications.

One can also modify the amino and/or carboxy termini of the peptidesdescribed herein. Terminal modifications are useful, to reducesusceptibility by proteinase digestion, and therefore can serve toprolong half life of the polypeptides in solution, particularly inbiological fluids where proteases may be present. Amino terminusmodifications include methylation (e.g., —NHCH₃ or —N(CH₃)₂),acetylation (e.g., with acetic acid or a halogenated derivative thereofsuch as α-chloroacetic acid, α-bromoacetic acid, or α-iodoacetic acid),adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminuswith any blocking group containing a carboxylate functionality definedby RCOO— or sulfonyl functionality defined by R—SO₂—, where R isselected from the group consisting of alkyl, aryl, heteroaryl, alkylaryl, and the like, and similar groups. One can also incorporate adesamino acid at the N-terminus (so that there is no N-terminal aminogroup) to decrease susceptibility to proteases or to restrict theconformation of the peptide compound. In certain embodiments, theN-terminus is acetylated with acetic acid or acetic anhydride.

Carboxy terminus modifications include replacing the free acid with acarboxamide group or forming a cyclic lactam at the carboxy terminus tointroduce structural constraints. One can also cyclize the peptidesdescribed herein, or incorporate a desamino or descarboxy residue at thetermini of the peptide, so that there is no terminal amino or carboxylgroup, to decrease susceptibility to proteases or to restrict theconformation of the peptide. Methods of circular peptide synthesis areknown in the art, for example, in U.S. Patent Application No.20090035814; Muralidharan and Muir, 2006, Nat Methods, 3:429-38; andLockless and Muir, 2009, Proc Natl Acad Sci USA. June 18, Epub.C-terminal functional groups of the peptides described herein includeamide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,and carboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof.

One can replace the naturally occurring side chains of the geneticallyencoded amino acids (or the stereoisomeric D amino acids) with otherside chains, for instance with groups such as alkyl, lower (C₁₋₆) alkyl,cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl amidedi(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower esterderivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocycles. Inparticular, proline analogues in which the ring size of the prolineresidue is changed from 5 members to 4, 6, or 7 members can be employed.Cyclic groups can be saturated or unsaturated, and if unsaturated, canbe aromatic or non-aromatic. Heterocyclic groups preferably contain oneor more nitrogen, oxygen, and/or sulfur heteroatoms. Examples of suchgroups include the furazanyl, furyl, imidazolidinyl, imidazolyl,imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino),oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g.,1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl(e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl,thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl groups.These heterocyclic groups can be substituted or unsubstituted. Where agroup is substituted, the substituent can be alkyl, alkoxy, halogen,oxygen, or substituted or unsubstituted phenyl.

One can also readily modify peptides by phosphorylation, and othermethods [e.g., as described in Hruby, et al. (1990) Biochem J.268:249-262].

The peptide compounds described herein also serve as structural modelsfor non-peptidic compounds with similar biological activity. Those ofskill in the art recognize that a variety of techniques are availablefor constructing compounds with the same or similar desired biologicalactivity as the prom-1 peptides, but with more favorable activity thanthe prom-1 peptide with respect to solubility, stability, andsusceptibility to hydrolysis and proteolysis [See, Morgan and Gainor(1989) Ann. Rep. Med. Chem. 24:243-252]. These techniques include, butare not limited to, replacing the peptide backbone with a backbonecomposed of phosphonates, amidates, carbamates, sulfonamides, secondaryamines, and N-methylamino acids. Further encompassed is a peptidemimetic of the peptide fragments of prom-1 described herein, includinge.g., peptidomimetics of SEQ. ID. Nos. 4-9. Such a peptidomimetic mayhave different amino acids from the peptide that it mimics butsubstantially retains the VEGF-binding, regenerative, pro-angiogenic,pro-cell proliferation, pro-cell migration, anti-angiogenic, anti-cellproliferation, pro-cell migration, pro-wound healing, or neuroprotectiveactivity of the peptide that it mimics.

Prom-1 Peptides in the Treatment of Disease

In one embodiment, provided herein is a composition comprising apharmaceutically acceptable carrier and a peptide fragment of a prom-1extracellular domain. In yet another embodiment, provided herein is acomposition comprising a pharmaceutically acceptable carrier and afusion protein comprising a peptide described herein or a conjugate of apeptide described herein. Also encompassed are compositions comprising avector carrying the coding nucleic acid for a fusion protein comprisinga peptide described herein or a polymer of peptides described herein.

In one embodiment, described herein is a method of promoting cellproliferation in a tissue in need thereof, the method comprisingcontacting the tissue with a composition comprising a peptide fragmentof a prom-1 extracellular domain having a conservative amino acidsubstitution variant thereof, or a conjugate or other derivativethereof.

In one embodiment, described herein is a method of promotingangiogenesis in a tissue in need thereof, the method comprisingcontacting the tissue with a composition comprising a peptide of aprom-1 extracellular domain or a variant or derivative thereof.

In one embodiment, the method of promoting angiogenesis in a tissue inneed thereof includes but is not limited to tissues that requirere-vascularization after disease and trauma. Re-vascularization isneeded for the rehabilitation of important organs, such as the heart,liver, bone, and lungs, after damage caused by disease and physicaltrauma (e.g., myocardial infarction, occlusive peripheral vasculardisease). Diseases that halt, block or reduce blood circulation include,but are not limited to, stroke, heart attack, myocardial ischemia,ischemic limbs, diabetes, vascular diseases such as peripheral vasculardisease (PVD), carotid artery disease, atherosclerosis, and renal arterydisease. Trauma such as those from car accidents and shock can result inreduced blood circulation to areas needing increased circulation duringthe healing process. In addition, treatment of a subject with a peptidedescribed herein is applicable to improving collateral coronary,peripheral artery, and carotid circulation in patients suffering fromimpaired wound healing, burn healing, bone fractures, neuropathy,impotence, erectile dysfunction, diabetic neuropathy, spinal cordinjury, nerve injury, and other vascular occlusive disorders such assickle cell disease, and stroke. As some of the prom-1 peptides canpotentiate the effect of VEGF on the vasculature, the peptides are alsocontemplated herein for use as vasodilators for the regulation of highblood pressure in a subject.

In one embodiment, the method of promoting angiogenesis is applied toerectile dysfunction, which can be caused by vascular disorders. The useof the prominin-1 peptides described herein can treat impotence byencouraging repair of the penile vascular network.

In one embodiment, the method of promoting cell proliferation and/orpromoting angiogenesis is applied in the context of wound healing, burnhealing, myocardial infarction, tissue repair, fertility, erectiledysfunction, cardiac hypertrophy, tissue grafts, and/or tissueengineered constructs. A variety of tissues, or organs comprisingorganized tissues, requiring angiogenesis include but are not limited tothe skin, muscle, gut, connective tissue, joints, bones and the liketypes of tissue in which blood vessels are required to nourish thetissue.

In one embodiment, the methods of promoting cell proliferation and/orpromoting angiogenesis further comprise contacting a tissue withadditional pro-angiogenic factors and/or growth promoting factors, e.g.,VEGF, FGF, PDGF, and IGF.

In one aspect, promoting angiogenesis can protect severely hypertrophiedhearts from ischemic injury. Myocardial hypertrophy is associated withprogressive contractile dysfunction, increased vulnerability toischemia-reperfusion injury, and is, therefore, a risk factor in cardiacsurgery. During the progression of hypertrophy, a mismatch developsbetween the number of capillaries and cardiomyocytes (heart musclecells) per unit area, indicating an increase in diffusion distance andthe potential for limited supply of oxygen and nutrients. Treatment ofhypertrophied hearts with VEGF resulted in an increase of microvasculardensity, improved tissue perfusion, and glucose delivery. (I. Friehs,et. al., 2004, The Annals of Thoracic Surgery, 77: 2004-2010). While notwishing to be bound by theory, the methods described herein forpromoting cell proliferation and/or promoting angiogenesis can addressthis mismatch by potentiating the effect of VEGF in increasing thecapillaries to improve the supply of nutrients to the cardiomyocytes.

In another aspect, promoting angiogenesis can stimulate bone repair andbone turnover. Several growth factors are known to be expressed in atemporal and spatial pattern during fracture repair. Exogenously addedVEGF enhances blood vessel formation, ossification, and new bonematuration (Street, J. et. al., 2002, PNAS, 99:9656-61). Accordingly,the method described herein for promoting cell proliferation and/orpromoting angiogenesis with prominin-1 peptide can be a therapy for bonerepair. Bone repair assays are provided herein (see section entitled“Bone Repair Assays”) to test the bone repair activity of pharmaceuticalcompositions comprising the peptides described herein.

In some aspects, the methods described herein for promoting cellproliferation and/or promoting angiogenesis are applicable to thetreatment of wounds, and particularly for the treatment of persistentwounds, such as diabetic ulcers. Wounds, in particular persistentwounds, which are difficult to heal, require a blood supply that cannourish the wound, mediate the healing process and minimize scarformation. Commonly used therapies for treating persistent wounds do notassist the wound to provide its own blood supply and therefore thehealing process remains slow. Persistent wounds can be ischemic wounds,for example, where the injury results from lack of oxygen due to poorcirculation such as in diabetes, scleroderma, and the like. Sclerodermais a disease involving an imbalance in tissue reformation giving rise tothe overproduction of collagen, and ultimately resulting in swelling andhardening of the skin (and affected organs). Diabetic wounds areespecially difficult to treat because the inadequate blood supply isoften complicated by other medical conditions such as peripheralvascular disease and neuropathy.

Agents such as the peptides described herein can be used to promotewound healing. A prominin-1 peptide used for wound healing will promotemore rapid wound closure and/or greater angiogenesis at a given timerelative to a similar wound not treated with the prom-1 peptide. Woundhealing assays are provided herein (see section entitled “Wound HealingAssays”) to test the wound healing activity of pharmaceuticalcompositions comprising the peptides described herein.

In one embodiment, the compositions described herein are administeredtopically to promote wound healing. In one embodiment, the peptidesdescribed herein are incorporated into a hydrogel or dressing or thelike for use in the treatment of wounds. Alternatively, the prom-1peptide compositions can be administered systemically. In otherembodiments, the prom-1 peptide compositions can be administereddirectly to the organ or tissue in need in the context of a scaffold orgel material, e.g., directly to a bone fracture site.

In another embodiment, the compositions described herein areadministered to the central nervous system to stimulate neuronal growth(e.g., neurite formation, axonal growth, axonal branching, and nervetropism).

In some aspects, the methods described herein for promoting cellproliferation and/or promoting angiogenesis can promote angiogenesis in3-D scaffold constructs of biodegradable polymeric scaffolds coated withthe peptides or engineered to contain cells expressing nucleic acidsencoding the peptide fragments. This equally applies to other scaffoldmaterials (such as hydroxylapatite and metals). The emergence of thetissue engineering (TE) field has resulted in the development of variousinterdisciplinary strategies primarily aimed at meeting the need toreplace organs and tissues lost due to diseases or trauma. In essence,the main TE approach is centered on seeding biodegradable scaffolds(both organic and inorganic such as poly(lactide-co-glycolide) andapatites) with donor cells, and optionally appropriate growth factor(s),followed by culturing and implantation of the scaffolds to induce anddirect the growth of new, functional tissue. The scaffold materialeventually disappears through biodegradation and is replaced by thespecific tissue. This scaffold-guided TE approach is aimed at creatingtissues such as skin, cartilage, bone, liver, heart, breast, etc.

Despite success with small (thin) tissue-engineered constructs, perhapsthe biggest roadblock in scaffold-guided TE is engineering large tissuevolumes. This challenge arises due to the lack of rapid vascularization(angiogenesis) of large three-dimensional (3-D) scaffold constructs.Accordingly, angiogenesis is a pre-requisite for scaffold-guided TE oflarge tissue volumes. Described herein is a method of promoting cellproliferation and/or promoting angiogenesis in a tissue-engineeredconstruct, the method comprising contacting the tissue construct with acomposition comprising a prom-1 peptide as that term is defined herein.

A number of biomolecules which induce or promote angiogenesis in tissueshave been identified. The most prominent of these are: growth factorssuch as vascular endothelial growth factor (VEGF), fibroblast growthfactor (FGF), epidermal growth factor (EGF), platelet-derived growthfactors (PDGFs) and transforming growth factors (TGFs); and nitric oxide(NO). Therefore, in one embodiment, the method of promoting cellproliferation and/or promoting angiogenesis in a tissue-engineeredconstruct further comprises administration of additional growth factorssuch as VEGF, FGF, EGF, PDGFs, TGFs, NO, and combinations thereof.

The patient treated according to the various embodiments describedherein is desirably a human patient, although it is to be understoodthat the principles of the invention indicate that the invention iseffective with respect to all mammals, which are intended to be includedin the term “patient”. In this context, a mammal is understood toinclude any mammalian species in which treatment of diseases associatedwith angiogenesis is desirable, particularly agricultural and domesticmammalian species.

In methods of treatment as described herein, the administration ofprom-1 peptides can be for either “prophylactic” or “therapeutic”purpose. When provided prophylactically, the prom-1 peptides, variants,fusion protein, polymer of peptides, conjugates, mimetics and/or codingnucleic acids are provided in advance of any symptom. The prophylacticadministration of the prom-1 peptides and/or coding nucleic acids servesto prevent or inhibit a disease or disorder associated with insufficientangiogenesis.

When provided therapeutically, a prom-1 peptide as described hereinand/or coding nucleic acid thereof is provided at (or after) the onsetof a symptom or indication of insufficient angiogenesis. Thus, theprom-1 peptides and/or coding nucleic acids can be provided either priorto the anticipated angiogenesis at a site or after the angiogenesis hasbegun at a site.

Angiogenesis Assays

Various methods of assaying for angiogenesis are described herein andreferenced below. The complete content of these references is herebyincorporated by reference. In general, to measure the pro-angiogenicactivity of an agent, e.g., a prominin-1 peptide as described herein,one will perform a given assay in the presence and absence of thepeptide composition. Further, where the prom-1 peptides described hereininteract with VEGF, it is preferred that the assays include VEGF (oranother pro-angiogenic factor) as the baseline or control assay as wellas in the prominin-1 containing assay.

Examples of well described angiogenesis assays that can be used to testor confirm pro-angiogenic activity of the peptides, variants, fusionproteins, peptidomimetics, peptide conjugates, or polymers of peptidesor other derivatives described herein include, but are not limited to invitro endothelial cell assays, rat aortic ring angiogenesis assays,cornea micro pocket assays (corneal neovascularization assays), andchick embryo chorioallantoic membrane assays (Erwin, A. et al. (2001)Seminars in Oncology 28(6):570-576).

Some examples of in vitro endothelial cell assays include methods formonitoring endothelial cell proliferation, cell migration, or tubeformation. It is anticipated that prominin-1 peptides as describedherein will affect each of these endothelial cell processes. Cellproliferation assays can use cell counting, BRdU incorporation,thymidine incorporation, or staining techniques (Montesano, R. (1992)Eur J Clin Invest 22:504-515; Montesano, R. (1986) Proc Natl. Acad. Sci.USA 83:7297-7301; Holmgren L. et al. (1995) Nature Med 1:149-153).

As one example of a cell proliferation assay, human umbilical veinendothelial cells are cultured in Medium 199 (Gibco BRL) supplementedwith 10% fetal bovine serum (Gibco BRL), 50 U/ml penicillin, 50 ng/mlstreptomycin, 2 mM L-glutamine and 1 ng/ml basic fibroblast growthfactor (bFGF) in T75 tissue culture flasks (Nunclon) in 5% CO₂ at 37° C.Cells are trypsinised (0.025% trypsin, 0.265 mM EDTA, GibcoBRL) andseeded in 96-well plates (Nunclon) at a density of 3000 cells/well/200μl and cultured for 3 days. Cells are starved in 1% serum for 24 hoursand are then treated with 1% serum containing 1 ng/ml bFGF in thepresence or absence of a pro-angiogenic agent for a further 48 hours.Two hours before the termination of incubation, 20 μl of CELLTITER 96®Aqueous One Solution Reagent (Promega Inc.) is added into each well.After the completion of incubation at 37° C. in a humidified, 5% CO₂atmosphere, the optical densities of the wells at 490 nm (“OD490”) arerecorded using a plate reader (Bio-Tek). The quantity of formazanproduct as measured by the amount of 490 nm absorbance is directlyproportional to the number of living cells in culture.

Alternatively, the incubation period of cells with the pro-angiogenicfactor can be allowed to proceed for up to 7 days. The cells are countedon a coulter counter on e.g., days 1, 3, 5 and 7. Remaining cells arefed by media replacement on these days. Data is plotted and doublingtime calculated using a regression analysis (cells in log phase ofgrowth). The doubling time for the cell is monitored as an indicator ofcell proliferative activity.

In cell migration assays, endothelial cells are plated on matrigel andmigration monitored upon addition of a chemoattractant (Homgren, L. etal. (1995) Nature Med 1:149-153; Albini, A. et al. (1987) Cancer Res.47:3239-3245; Hu, G. et al. (1994) Proc Natl Acad Sci USA 6:12096-12100;Alessandri, G. et al. (1983) Cancer Res. 43:1790-1797.)

Another migration assay monitors the migration of bovine aorticendothelial cells. In the assay, bovine aortic endothelial (BAE) cellsare allowed to grow to confluence in Dulbecco's modified Eagle medium(DMEM, GibcoBRL) containing 10% fetal bovine serum (GibcoBRL) in 12-wellplates (Nunclon). The monolayers are then ‘wounded’ by scraping adisposable pipette tip across the dishes. After washing with Dulbecco'sPBS plus calcium (0.1 g/L) (GIBCO™, Invitrogen Corporation), the woundedmonolayers are cultured for a further 48 hours in fresh 1% serum in thepresence or absence of a pro-angiogenic agent.

The degree of movement of cells in the wounded mono layers is determinedby taking photomicrographs at the time of the initial wounding and 48hours after wounding. The photomicrographs are taken at 20×magnification, e.g., on an Olympus CK2 inverted microscope and printedto a standard size of 15 cm wide by 10 cm deep. A grid with lines 1.5 cmapart and 10 cm long running parallel to a baseline is placed over thephotograph. The baseline is placed on the “wounding line” above whichthe cells have originally been scraped off. The number of cellsintercepted by each of the lines is recorded. This allows an assessmentof the number of cells that have migrated 1.5, 3.0, 4.5, 6.0, 7.5 or 9.0cm away from the baseline on the photomicrograph.

Endothelial tube formation assays monitor vessel formation (Kohn, E C.et al. (1995) Proc Natl Acad Sci USA 92:1307-1311; Schnaper, H W. et al.(1995) J Cell Physiol 165:107-118).

Rat aortic ring assays have been used successfully for the evaluation ofangiogenesis drugs (Zhu, W H. et al., (2000) Lab Invest 80:545-555;Kruger, E A. et al., (2000) Invasion Metastas 18:209-218; Kruger, E A.et al., (2000) Biochem Biophys Res Commun 268:183-191; Bauer, K S. etal. (1998) Biochem Pharmacol 55:1827-1834; Bauer, K S. et al., (2000) JPharmacol Exp Ther 292:31-37; Berger, A C. et al., (2000) Microvasc Res60:70-80.). Briefly, the assay is an ex vivo model of explant rat aorticring cultures in a three dimensional matrix. One can visually observeeither the presence or absence of microvessel outgrowths. The humansaphenous angiogenesis assay, another ex vivo assay, can also be used(Kruger, E A. et al. (2000) Biochem Biophys Res Commun 268:183-191).

Another common angiogenesis assay is the corneal micropocket assay(Gimbrone, M A. et al., (1974) J Natl Canc Inst. 52:413-427; Kenyon, BM. et al., (1996) Invest Opthalmol V is Sci 37:1625-1632; Kenyon, B M.et al., (1997) Exp Eye Res 64:971-978; Proia, A D. et al., (1993) ExpEye Res 57:693-698). Briefly, neovascularization into an avascular spaceis monitored in vivo. This assay is commonly performed in rabbit, rat,or mouse.

The chick embryo chorioallantoic membrane assay has been used often tostudy tumor angiogenesis, angiogenic factors, and antiangiogeniccompounds (Knighton, D. et al. (1977) Br J Cancer 35:347-356; Auerbach,R. et al. (1974) Dev Biol 41:391-394; Ausprunk, D H. et al. (1974) DevBiol 38:237-248; Nguyen, M. et al. (1994) Microvasc Res 47:31-40). Thisassay uses fertilized eggs and monitors the formation of primitive bloodvessels that form in the allantois, an extra-embryonic membrane. Thisassay functions as an in vivo endothelial cell proliferation assay.

Other in vivo angiogenesis assays are described in U.S. Pat. No.5,382,514 and the directed in vivo angiogenesis assay (DIVAA™) systemmade by Trevigen, Inc. In these assays, a pro-angiogenic factor isincorporated into a tissue compatible matrix or hydrogel material suchas Matrigel (GibcoBDL) or in the angioreactor Cultrex® DIVAA™, thematrix material or angioreactor is implanted subdermally into nude mice.Over time, usually days, microvessels invade the matrix material orangioreactor. The matrix material or angioreactor are then excised fromthe host mouse and examined.

Wound Healing Assays

The prominin-1 peptides described herein can be used to facilitate,enhance or accelerate wound healing. Wound healing, or wound repair, isan intricate process in which the skin (or some other organ) repairsitself after injury. The classic model of wound healing is divided intofour sequential, yet overlapping, phases: (1) hemostasis, (2)inflammatory, (3) proliferative and (4) remodeling. Angiogenesis occursduring the proliferative phase of wound healing and promotes woundcontraction (i.e., a decrease in the size of the wound). Microvascularin-growth into damaged tissue is an essential component of the normalhealing process. In fact, wound therapy is often aimed at promotingneovascularization.

Thus, a wound healing assay can be used as an angiogenesis assay toassess the effect of a given prom-1 peptide, variant, or derivativedescribed herein. Such wound healing assays include, but are not limitedto, ear punch assays and full thickness dorsal skin assays. Woundhealing assays can be performed as described in U.S. PublishedApplication No. 20060147415, entitled “Composition and method fortreating occlusive vascular diseases, nerve regeneration and woundhealing,” which is incorporated herein by reference in its entirety. Theterm “full thickness” is used herein to describe a wound that includesthe epidermal layer and at least a portion of the dermal layer. The term“full thickness” also encompasses a deep wound to the level of thepanniculus carnosus that removes epidermal, dermal, subcutaneous, andfascia layers.

Full thickness dorsal skin wounding assays can be performed as describedin e.g., Luckett-Chastain, L R and Galluci, R M, Br J Dermatol. (2009)Apr. 29; Shaterian, A et al., Burns (2009) May 5; Lee, W R, et al.,Wound Repair Regen (2009) Jun. 12; and Safer, J D, et al., Endocrinology(2005) 146(10):4425-30, which are herein incorporated by reference intheir entirety. Dorsal skin wounding assays can be performed using rator mouse models.

Whereas, the ear punch wound assay is used to generate the wound healingdata described herein, it is expected that any of the other woundhealing assays described herein will provide similar or superior resultswith prom-1 peptides as described. In one embodiment, a full-thicknesswound is effected by removing a section of skin (e.g., 1.5 mm diameter)from the dorsal surface (e.g., back) of an anesthetized animal by e.g.,surgical incision. If so desired, the section of skin to be wounded canbe pre-treated with a candidate pro-angiogenic factor prior to woundinduction by e.g., subcutaneous injection. Alternatively, the wound canbe treated using a candidate pro-angiogenic factor coincident with orimmediately following wounding using methods known to one of skill inthe art. The size, area, rate of healing, contraction and histology ofthe wound are assessed at different time points by methods known tothose of skill in the art. The wound size of an animal is assessed bymeasuring the unclosed wound area compared to the original wound area.Wound healing can be expressed as either percent wound closure orpercent wound closure rate. Wounds can be harvested at different timepoints by euthanizing the animal and removing a section of skinsurrounding the wound site for histological analysis if so desired.

The capacity of a candidate pro-angiogenic factor to induce oraccelerate a healing process of a skin wound can be determined byadministering the candidate pro-angiogenic factor to skin cellscolonizing the damaged skin or skin wound area and evaluating thetreated damaged skin or wounds for e.g., angiogenesis and/or epidermalclosure and/or wound contraction. As known to those of skill in the art,different administration methods (e.g., injection or topicaladministration) can be used to treat the skin wound, and differentconcentrations of the candidate pro-angiogenic factor can be tested. Astatistically significant increase in the incidence of vessel formationand/or epidermal closure and/or wound contraction, over an untreatedcontrol, indicates that a tested candidate pro-angiogenic factor iscapable of inducing or accelerating a healing process of a damaged skinor skin wound. Positive results are indicated by a reduction in thepercent wound area of a mouse treated with a candidate pro-angiogenicfactor of at least 5% compared to the wound area of an untreated orvehicle treated mouse at the same timepoint; preferably the reduction inpercent wound area is at least 7%, at least 8%, at least 9%, at least10%, at least 12%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99%, or even100% (i.e., wound is completely closed).

An ear punch model can also be used to assess rates of angiogenesis orwound healing, in a design similar to that for the full thickness dorsalback skin assay. The model consists of wounding the ear of an animalusing a circular punch of a standard size (e.g., 2.25 mm). The wound istreated daily with either a matrigel vehicle or a matrigel containing acandidate pro-angiogenic factor. This assay is further described hereinin the Examples section.

Neurodegenerative Diseases

VEGF has neurotrophic and neuroprotective effects. Given the effects ofprom-1 peptides as described herein on VEGF-mediated angiogenesis, andparallels between VEGF's pro-angiogenic and neurotrophic activities, itis anticipated that prom-1 peptides as described herein can potentiateor enhance the neurotrophic or neuroprotective effects of VEGF. Theprom-1 peptides can be used to treat neurodegenerative diseases asdescribed in e.g., U.S. Published Application No. 20060147415, entitled“Composition and method for treating occlusive vascular diseases, nerveregeneration and wound healing,” which is incorporated herein byreference in its entirety. Furthermore, the prom-1 peptides describedherein can be used to stimulate neuronal growth, axonal elongation,axonal branching and neurite formation.

In one embodiment, the prom-1 peptide compositions described herein areused as neuroprotective agents to prevent and/or treat diseasesassociated with neurodegeneration or nerve damage. Thus, in oneembodiment, provided herein is a method of promoting neuroprotection,the method comprising contacting a neuronal cell with an isolated prom-1peptide of a prominin polypeptide, wherein the peptide binds VEGF, andwherein the contacting promotes neuroprotection of the neuronal cell. Inanother embodiment, the contacting of the neuronal cell prevents ordelays neuronal cell death relative to neuronal cell death occurring inthe absence of such contacting, or wherein such contacting promotesnerve regeneration by stimulating neuronal growth.

Neurodegeneration or nerve damage can result from e.g., stroke, heatstress, head and spinal cord trauma, and bleeding that occurs in thebrain, the pressure from which eventually causes the death of one ormore neurons; often neuronal death begins long before the patient willever experience any symptoms.

Neurodegeneration can also be a result of neurodegenerative diseasescaused by the deterioration of neurons, which over time leads tophysical manifestations. Neurodegenerative diseases of the centralnervous system include e.g., intracerebral hemorrhage (ICH),neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease and other degenerative diseases of the basal ganglia; otherneurological causes of memory loss or impairment, including Down'ssyndrome, Creutzfeldt-Jakob disease, prion diseases, cerebral ischemiaand stroke; multiple sclerosis; motor neuron disease, such as amyotropiclateral sclerosis; neurological viral disease; post-surgicalneurological dysfunction; Huntington's disease; hereditary spastichemiplegia; primary lateral sclerosis; spinal muscular atrophy;Kennedy's disease; Shy-Drager syndrome; Progressive Supranuclear Palsy;Lewy Body Disease; neuronopathies; dementia; frontotemporal lobedementia; ischemic disorders (e.g., cerebral or spinal cord infarctionand ischemia, chronic ischemic brain disease, and stroke); kaumas (e.g.,caused by physical injury or surgery, and compression injuries);affective disorders (e.g., stress, depression and post-traumaticdepression); neuropsychiatric disorders (e.g., schizophrenia, multiplesclerosis, and epilepsy); learning and memory disorders; trigeminalneuralgia; glossopharyngeal neuralgia; Bell's Palsy; myasthenia gravis;progressive muscular atrophy; progressive bulbar inherited muscularatrophy; herniated, ruptured and prolapsed vertebrate disk syndromes;cervical spondylosis; plexus disorders; thoracic outlet destructionsyndromes; peripheral neuropathies; prophyria; muscular dystrophy; apolyglutamine repeat disease; and spongiform encephalopathy. Ocularneuron disorders can also be treated with peptides as described hereinand include, but are not limited to, retina or optic nerve disorders;optic nerve damage and optic neuropathies such as Lebers hereditaryoptic neuropathy, autosomal dominant optic atrophy, optic neuritis;disorders of the optic nerve or visual pathways; toxic neuropathies andtoxic retinopathies; optic atrophy; glaucoma; retinal degenerations suchas retinitis pigmentosa, macular degeneration, and diabetic retinopathy.

Neuroprotective effects of peptide compositions as described herein canbe measured using in vivo or in vitro assays that assess e.g., neuronsurvival, neuron growth, neurite production, re-innervation, improvedbehavioral symptoms etc. As one example, in vitro assays of neuriteoutgrowth can be used to evaluate or monitor the neuroprotective effectsof peptide compositions as described herein. In vitro assays of neuritegrowth are well known in the art and are described in, for example, Jinand Strittmatter, J Neurosci 17:6256-6263 (1997); Fournier et al.,Methods Enzymol. 325:473-482 (2000); Zheng et al., Neuron 38:213-224(2003); Wang et al., Nature 417:941-944 (2002), and Neumann et al.,Neuron 34:885-893 (2002), which are all incorporated herein by referencein their entirety. Kits for measuring and quantifying neurite outgrowthare commercially available from e.g., Chemicon (Billerica, Mass.),Millipore (Billerica, Mass.), and Thermo Scientific Pierce ProteinResearch Products (Rockford, Ill.). Thus, for example, CHEMICON'sNeurite Outgrowth Assay Kit (Catalog number NS200) uses microporousfilter technology for the quantitative testing of compounds thatinfluence neurite formation and repulsion. With this system, it ispossible to analyze biological and pharmacological agentssimultaneously, directly evaluate adhesion and guidance receptorfunctions responsible for neurite extension and repulsion, as well asthe analysis of gene function in transfected cells. The microporousfilter allows for biochemical separation and purification of neuritesand cell bodies for detailed molecular analysis of protein expression,signal transduction processes and identification of drug targets thatregulate neurite outgrowth or retraction processes.

In one embodiment, an in vitro neurite cell-based assay involvesculturing neuronal cells in the presence and absence of a candidateneuroprotective agent (e.g., a peptide composition as described herein)and determining the change in neurite length. The agent is identified orconfirmed as neuroprotective when the neurite length is longer in thepresence of the candidate agent than the length of a neurite inuntreated cells. Assays will generally be performed in the presence ofVEGF, plus and minus a prom-1 peptide composition. In such a cell-basedassay, the neuronal cells can be primary neurons, or can, for example,be derived from cells or cell lines, including stem cells, e.g.,embryonic stem (ES) cells. In other embodiments, the neurons can, forexample, be selected from the group consisting of cerebellar granuleneurons, dorsal root ganglion neurons, and cortical neurons. In atypical protocol, primary neurons isolated from rodent neural tissue(including cerebellar granule neurons, dorsal root ganglion neurons, andcortical neurons) are cultured on 96-well tissue culture dishes coatedwith immobilized whole myelin or myelin associated proteins (e.g.,Nogo66, MAG and/or OMgp). Following a defined time in culture, typically24-48 hours, the neurons are fixed with 4% paraformaldehyde and stainedwith a neuronal marker (anti-class III b-Tubulin, Covance). Imageacquisition and analysis are then performed using e.g., the ImageXpressautomated imaging system (Molecular Devices). Data are analyzed forchanges in maximal or total neurite length per neuron. An enhancedneurite growth response in the presence of a prom-1 peptide as describedherein confirms the neurotrophic and/or neuroprotective effects of thegiven prom-1 peptide (see Example 10).

In vivo assays are known to those of skill in the art and include animalmodels of various neurodegenerative diseases, such as spinal cord injurymodels, visual cortex plasticity models, and other models known in theart. Thus, regeneration and plasticity can be studied in models ofplasticity following unilateral pyramidotomy and models of traumaticbrain injury. Other models of neurodegeneration include mouse models ofmultiple sclerosis, such as experimental autoimmune encephalitis (EAE),models of amyotrophic lateral sclerosis (ALS), such as the SODI mutantmouse, transgenic animal models of Alzheimer's disease, and animalmodels of Parkinson's. The beneficial effect of prom-1 peptideadministration can be evaluated or confirmed in any of these assays,e.g., by administering VEGF alone or VEGF and a prom-1 peptide asdescribed herein. Alternatively, it is contemplated that prom-1 peptidescan act in such assays without exogenously added VEGF or otherpro-angiogenic or neurotrophic agents.

In one embodiment, neuronal regeneration is assessed by measuring axonalregeneration in a model of optic nerve crushing (Fischer D, et al., J.Neurosci. 18, 1646 (2004)). In a typical protocol, adult mouse opticnerves are exposed behind the eyeball and crushed. Immediately afterinjury in adult mice, Gelfoam soaked in a solution containing a peptidedescribed herein or vehicle control is placed against the crush site ofthe nerve and replaced every three days for the first six days. Animalsare sacrificed two weeks post injury followed by transcardial perfusionwith 4% paraformaldehyde. Optic nerves are cryosectioned at 10 μm andstained with an anti-GAP43 antibody (Chemicon) to detect regeneratingaxons.

Other in vivo assays include assessing behavioral changes in peptidetreated and untreated animal models of neurodegenerative disease. Forexample, akinesia is measured by noting the latency in seconds (s) ofthe animals to move all four limbs. In a typical protocol, each animalis initially acclimatized for 5 min on a wooden elevated platform (40cm×40 cm×30 cm) used for measuring akinesia in mice. Using a stopwatch,the time taken (s) by the animal to move all four limbs is recorded. Ingeneral, measurement is stopped once the latency period reaches 180 sec.Another behavioral measure is catalepsy, which refers to the inabilityof an animal to correct an externally imposed posture. In oneembodiment, catalepsy is measured by placing the animal on a flathorizontal surface with both the hind limbs on a square wooden block (3cm high) and the latency in seconds is measured to move the hind limbsfrom the block to the ground. A swim test is also used for measuring theextent of neurodegeneration in an animal model. Swim-tests are carriedout following treatment of the animals with a peptide and are performedin water tubs. In a typical protocol, the depth of water is about 12 cmand the temperature maintained at around 27° C. Animals are scored forswim tests according to the following scale: 0, hind part sinks withhead floating; 1, occasional swimming using hind limbs while floating onone side; 2, occasional floating/swimming only; 3, continuous swimming(see Haobam et al. (2005) Behay. Brain Res. 163, 159-167). Thebeneficial neuroprotective or neurotrophic effects of prom-1 peptidesdescribed herein would be confirmed by a statistically significantimprovement in any of the behaviors monitored by these assays.

Clinically, an effective dose of a peptide described herein, oreffective regimen, is a combination of dose and dosing that provides foran improvement in the symptoms associated with the particular neuronalor neurodegenerative disease, e.g., Parkinson's disease as assessed bythe United Parkinson's Disease Rating Scale (UPDRS), or the use ofsurrogate markers. For example, the motor abilities of a Parkinson'spatient may improve, where motor symptoms may include motorfluctuations, dyskinesias, off-period dystonia, freezing, and falls.Alternatively, improvement may be assessed by imaging, e.g., bymonitoring of dopamine uptake, or striatal neuron function. The standardtool for tracking Parkinson's disease progress and response to therapyis the United Parkinson's Disease Rating Scale (UPDRS). The UPDRS issubdivided into three scales including cognitive and mood aspects, motoraspects, and activities of daily living (ADL). A lower score indicates abetter condition than a higher score. The UPDRS is readily available,e.g., see Fahn S, Elton R, Members of the UPDRS Development Committee,In: Fahn S, Marsden C D, Caine D B, Goldstein M, eds. RecentDevelopments in Parkinson's Disease, Vol 2. Florham Park, N.J. MacmillanHealth Care Information 1987, pp 15 3-163, 293-304.

Further clinical tests for assessing neuroprotection can be used in theclinical setting by those of skill in the art of medicine. The treatmentof a neurodegeneration as a result of brain injury can be monitored byemploying a variety of neurological measurements. For example, atherapeutic response can be monitored by determining if, for example,there is an improvement in the subject's: a) maximum daily Glasgow ComaScore; b) duration of coma; 3) daily intracranial pressure(ICP)—therapeutic intensity levels; 4) extent of cerebral edema/masseffect measured on serial CT scans; and, 5) duration of ventilatorsupport. A brief description of each of these measurements is providedbelow.

The Glasgow Coma Score (index GCS) is a reflection of the depth ofimpaired consciousness and is best obtained following initialresuscitation (oxygenation, rehydration and support of blood pressure)but prior to use of sedating drugs, neuromuscular blocking agents, orintubation.

The ICP of patients with severe brain injury is often monitored with anintracranial pressure device. Monitoring ICP can provide a measure ofcerebral edema. However, inherent variability and analysis complexitiesdue to therapeutic intervention exist. To adjust for these interventionsa therapeutic intensity scale was developed. This scale, known as theTherapeutic Intensity Level (TIL), measures treatment aggressiveness forelevated ICPs (Allolio et al. (1995) European Journal of Endocrinology133(6): 696-700; Adashi et al. (1996) Reproductive endocrinology,surgery, and technology Philadelphia: Lippincott-Raven; and, Beers etal. eds. (1999) The Merck Manual of Diagnosis and Therapy. 17th ed.,Merck Sharp & Dohme Research Laboratories, Rahway, N.J.).

The extent of cerebral edema and mass effect can be determined by CTscans. For example, the volume of focal lesions can be measured. Masslesions, either high-density or mixed-density abnormalities, areevaluated by measuring the area of the abnormality as a region ofinterest, multiplying the area by the slice thickness, and summing thesevolumes for contiguous slices showing the same lesion. Each lesion ismeasured three times, and the mean volume entered. This technique hasbeen shown to be reliable (Garcia-Estrada et al. (1993) Brain Res628(1-2): 271-8). Intracerebral lesions can be further characterized bylocation (frontal, temporal, parietal, occipital, basal ganglia, or anycombination).

In addition to the neurological measurements discussed above, atherapeutic response can also be assayed through various functional andneuropsychological outcomes. Several standardized measures ofneuropsychological and functional performance are known. For instancesubjects may display an improvement in the Glasgow Outcome Scale(GOS)/Glasgow Outcome Scale Extender (GOSE) and/or in the DisabilityRating Scale (DRS). The Glasgow Outcome Score is one of the most widelyused measures of brain injury recovery in the world (Garcia-Estrada etal. (1999) Int J Dev Neurosci 17(2): p. 145-51). Patients are classifiedinto one of five categories: death, persistent vegetative state, severedisability, moderate disability, and good recovery. It is easy toadminister and score, and has a high degree of reliability and validity.The Disability Rating Scale (DRS) offers more precision than the GOS formeasuring outcomes of moderate brain injury (Goodman et al. (1996) JNeurochem 66(5): 1836-44). The DRS consists of an eight-item rating ofarousal and awareness, daily living activities, physical dependence, andemployability (Vedder et al. (1999) J Neurochem 72(6):2531-8).Inter-rater reliability for the entire DRS is high (0.97 to 0.98).

The Functional Independence Measure (FIM) can be used to assess physicaland cognitive disability. It contains 18 items in the following domains:self-care, sphincter control, mobility, locomotion, communication, andsocial cognition (Baulieu (1997) Mult Scler 3(2):105-12). The FIM hasdemonstrated reliability and validity as an outcome measure followingmoderate and severe TBI (Jung-Testas et al. (1994) J Steroid Biochem MolBiol 48(1):145-54).

The Sickness Impact Profile is one method for measuring self-perceivedhealth status (Schumacher et al. (1995) Ciba Found Symp 191: p. 90-112and Koenig et al. (1995) Science 268(5216):1500-3). It consists of 136questions divided into 12 categories: sleep and rest, eating, work, homemanagement, recreation and pastimes, ambulation, mobility, body care andmovement, social interaction, alertness, behavior, emotional behavior,and communication. It has been widely used across a variety of diseasesand injuries, including head injury (Thomas et al. (1999) Spine24:2134-8). Baseline SIP scores will reflect pre-injury health status,while follow-up scores will examine post-injury functioning.

In one embodiment, the compositions described herein are administered tothe central nervous system to stimulate neuronal growth (e.g., neuriteformation, axonal growth, axonal branching, and nerve tropism).

Bone Repair Assays

Method 1: Each mouse is anesthetized with a ketamine/xylazine anestheticand an incision is made over the anteromedial surface of the righttibial diaphysis. The muscle is blunt dissected to expose the periostealsurface and a 0.6 mm diameter penetrating hole is created in the medialcortex approximately 1 mm distal from the termination of the tibialtuberosity. Following surgery and/or treatment with the peptidesdescribed herein, all animals undergo high resolution micro-CT scan(Scanco vivaCT 40; 11 μm voxel resolution) to confirm the fracture. Asecond and third micro-CT scan are performed in all animals at 12 and 21days, respectively to monitor the progress of bone healing and provide aquantitative analysis of the bone mineral density at the fracture site.

Method 2: Each mouse is anesthetized with a ketamine/xylazine anestheticand a small incision is made on the dorsolateral side of the thigh andextended over the knee region. A longitudinal incision is made in thepatellar tendon, and a 0.5 mm hole is drilled above the tibiatuberosity. A fracture is then made by cutting the shaft of tibia. Afracture generated in this manner is known to heal through bothendochondral and intramembranous ossification.

Alternatively, the calvarial bone repair assay describe in Example 12can be used.

Peptides described herein are mixed with MATRIGEL™ and injected into thefracture site using a microsyringe. The animals are allowed free,unrestricted weight bearing in cages after recovery from anesthesia. Atdifferent time points (3, 4, 7, 14, and 21 d) after the fracture, thebone mineral density at the fracture site is analyzed using a SmallAnimal Bone Densitometer.

Pro-Angiogenic Factors

Pro-angiogenic factors are factors that directly or indirectly promotenew blood vessel formation. These factors can be expressed and secretedby normal and tumor cells. Pro-angiogenic factors derived from prom-1 asdescribed can be administered in combination with other pro-angiogenicfactors including, but not limited to, EGF, E-cadherin, VEGF(particularly VEGF isoforms: VEGF 121, 145 and 165), angiogenin,angiopoietin-1, fibroblast growth factors: acidic (aFGF) and basic(bFGF), fibrinogen, fibronectin, heparanase, hepatocyte growth factor(HGF), insulin-like growth factor-1 (IGF-1), IGF, BP-3, PDGF, VEGF-AVEGF-C, pigment epithelium-derived factor (PEDF), vitronection, leptin,trefoil peptides (TFFs), CYR61 (CCN1) and NOV (CCN3), leptin, midkine,placental growth factor platelet-derived endothelial cell growth factor(PD-ECGF), platelet-derived growth factor-BB (PDGF-BB), pleiotrophin(PTN), progranulin, proliferin, transforming growth factor-alpha(TGF-alpha), transforming growth factor-beta (TGF-beta), tumor necrosisfactor-alpha (TNF-alpha), c-Myc, granulocyte colony-stimulating factor(G-CSF), stromal derived factor 1 (SDF-1), scatter factor (SF),osteopontin, stem cell factor (SCF), matrix metalloproteinases (MMPs),thrombospondin-1 (TSP-1), and inflammatory cytokines and chemokines thatare inducers of angiogenesis and increased vascularity, eg. CCL2(MCP-1), interleukin-8 (IL-8) and CCL5 (RANTES).

Synthesis of Peptides

Prom-1 peptides, including variants and derivatives thereof can bechemically synthesized and purified by biochemical methods that are wellknown in the art such as solid phase peptide synthesis using t-Boc(tert-butyloxycarbonyl) or FMOC (9-flourenylmethloxycarbonyl) protectiongroup described in “Peptide synthesis and applications” in Methods inmolecular biology Vol. 298, Ed. by John Howl and “Chemistry of PeptideSynthesis” by N. Leo Benoiton, 2005, CRC Press, (ISBN-13:978-1574444544) and “Chemical Approaches to the Synthesis of Peptidesand Proteins” by P. Lloyd-Williams, et. al., 1997, CRC-Press, (ISBN-13:978-0849391422), Methods in Enzymology, Volume 289: Solid-Phase PeptideSynthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), AcademicPress; 1st edition (1997) (ISBN-13: 978-0121821906); U.S. Pat. Nos.4,965,343, and 5,849,954 and these are all hereby incorporated byreference in their entirety.

Solid phase peptide synthesis, developed by R. B. Merrifield, 1963, J.Am. Chem. Soc. 85 (14): 2149-2154, was a major breakthrough allowing forthe chemical synthesis of peptides and small proteins. An insolublepolymer support (resin) is used to anchor the peptide chain as eachadditional alpha-amino acid is attached. This polymer support isconstructed of 20-50 μm diameter particles which are chemically inert tothe reagents and solvents used in solid phase peptide synthesis. Theseparticles swell extensively in solvents, which makes the linker armsmore accessible.

Organic linkers attached to the polymer support activate the resin sitesand strengthen the bond between the alpha-amino acid and the polymersupport. Chloromethyl linkers, which were developed first, have beenfound to be unsatisfactory for longer peptides due to a decrease in stepyields. The PAM (phenylacetamidomethyl) resin, because of the electronwithdrawing power of the acid amide group on the phenylene ring,provides a much more stable bond than the classical resin. Anotheralternative resin for peptides under typical peptide synthesisconditions is the Wang resin. This resin is generally used with the FMOClabile protecting group.

A labile group protects the alpha-amino group of the amino acid. Thisgroup is easily removed after each coupling reaction so that the nextalpha-amino protected amino acid may be added. Typical labile protectinggroups include t-Boc (tert-butyloxycarbonyl) and FMOC. t-Boc is a verysatisfactory labile group which is stable at room temperature and easilyremoved with dilute solutions of trifluoroacetic acid (TFA) anddichloromethane. FMOC is a base labile protecting group which is easilyremoved by concentrated solutions of amines (usually 20-55% piperidinein N-methylpyrrolidone). When using FMOC alpha-amino acids, an acidlabile (or base stable) resin, such as an ether resin, is desired.

The stable blocking group protects the reactive functional group of anamino acid and prevents formation of complicated secondary chains. Thisblocking group must remain attached throughout the synthesis and may beremoved after completion of synthesis. When choosing a stable blockinggroup, the labile protecting group and the cleavage procedure to be usedshould be considered.

After generation of the resin bound synthetic peptide, the stableblocking groups are removed and the peptide is cleaved from the resin toproduce a “free” peptide. In general, the stable blocking groups andorganic linkers are labile to strong acids such as TFA. After thepeptide is cleaved from the resin, the resin is washed away and thepeptide is extracted with ether to remove unwanted materials such as thescavengers used in the cleavage reaction. The peptide is then frozen andlyophilized to produce the solid peptide. This is generally thencharacterized by HPLC and MALDI before being used. In addition, thepeptide should be purified by HPLC to higher purity before use.

Commercial peptide synthesizing machines are available for solid phasepeptide synthesis. For example, the Advanced Chemtech Model 396 MultiplePeptide Synthesizer and an Applied Biosystems Model 432A Peptidesynthesizer are suitable. There are commercial companies that makecustom synthetic peptides to order, e.g., Abbiotec, Abgent, AnaSpecGlobal Peptide Services, LLC. Invitrogen and rPeptide, LLC.

The prom-1 peptides and derivatives thereof can also be synthesized andpurified by molecular methods that are well known in the art. Forexample, recombinant protein may be expressed in bacteria, mammal,insect, yeast, or plant cells.

Conventional polymerase chain reaction (PCR) cloning techniques can beused to clone a nucleic acid encoding a prom-1 peptide, using the mRNAof the intact full length prom-1 as the template for PCR Cloning.Alternatively, the sense and anti-sense strand of the coding nucleicacid can be made synthetically and then annealed together to form thedouble-stranded coding nucleic acid. Ideally, restriction enzymedigestion recognition sites should be designed at the ends of the senseand anti-sense strand to facilitate ligation into a cloning vector orother vectors. Alternatively, a 3′A overhang can be include for thepurpose of TA-cloning that is well known in the art. Such coding nucleicacids with 3′A overhangs can be easily ligated into the Invitrogentopoisomerase-assisted TA vectors such as pCR®-TOPO, pCR®-Blunt II-TOPO,pENTR/D-TOPO®, and pENTR/SD/D-TOPO®. The coding nucleic acid can becloned into a general purpose cloning vector such as pUC19, pBR322,pBluescript vectors (Stratagene Inc.) or pCR TOPO® from Invitrogen Inc.The resultant recombinant vector carrying the nucleic acid encoding aprom-1 peptide can then be used for further molecular biologicalmanipulations such as site-directed mutagenesis for variant prom-1peptide and/or to reduce the immunogenic properties of the peptide orimprove protein expression in heterologous expression systems, or can besubcloned into protein expression vectors or viral vectors for thesynthesis of fusion protein comprising prom-1 peptides and proteinsynthesis in a variety of protein expression systems using host cellsselected from the group consisting of mammalian cell lines, insect celllines, yeast, bacteria, and plant cells.

In one embodiment, the invention provides cells engineered to expressnucleic acids encoding peptides of the invention. Preferably, the cellsare eukaryotic cells. The nucleic acids are operationally linked to apromoter. The expression construct can further comprise a secretorysequence to assist purificationof the peptide from the cell culturemedium.

In one embodiment, a sense nucleic acid encoding LCGNSFSGGQPS (SEQ IDNO: 4) is 5′ CTGTGCGGCAACAGCTTTAGCGGCGGCCAGCCGAGC 3′(SEQ. ID. No. 10)and the complementary anti-sense nucleic acid is 5′ isGCTCGGCTGGCCGCCGCTAAAGCTGTTGCCGCACAG 3′(SEQ. ID. No. 11).

PCR amplified coding nucleic acids or annealed sense and anti-sensenucleic acid with 3′A overhang can cloned into a vector using the TOPO®cloning method in Invitrogen topoisomerase-assisted TA vectors such aspCR®-TOPO, pCR®-Blunt II-TOPO, pENTR/D-TOPO®, and pENTR/SD/D-TOPO®. BothpENTR/D-TOPO®, and pENTR/SD/D-TOPO® are directional TOPO entry vectorswhich allow the cloning of the DNA sequence in the 5′→3′ orientationinto a Gateway® expression vector. Directional cloning in the 5′→3′orientation facilitate the unidirectional insertion of the DNA sequenceinto a protein expression vector such that the promoter is upstream ofthe 5′ ATG start codon of the nucleic acid, thus enablingpromoter-driven protein expression. The recombinant vector carrying aprom-1 peptide coding nucleic acid can be transfected into andpropagated in a general cloning E. coli cells such as XL1Blue, SURE(Stratagene) and TOP-10 cells (Invitrogen).

It is envisioned that multiple copies of the nucleic acid encodingprom-1 peptide can be ligated in tandem such that a polymer of prom-1peptides can be expressed. Protease cleavage sites can be designed andincluded between the nucleic acid to facilitate liberation of eachprom-1 peptide from the polymeric prom-1 peptide if so desired. Examplesof protease cleavage sites include but are not limited to those ofenterokinase, chymotrypsin, and thrombin.

Methods of making conservative amino acid substitutions are also wellknown to one skilled in the art and include but are not limited tosite-specific mutagenesis using oligonucleotide primers and polymerasechain reactions. A conservative substitution variant of a prom-1 peptideof 12 or fewer amino acids (as a non-limiting example, the #237 prom-1peptide) described herein can have 1 to 4 conservative amino acidsubstitutions but will, as will all prom-1 peptide variants orderivatives, substantially retain VEGF binding activity. In oneembodiment, the 1-4 substitutions are not located within the 6C-terminal amino acids of the #237 peptide. In another embodiment, thesubstitutions do not change the C-terminal alanine and valine aminoacids of the #237 peptide. Variant prom-1 peptides can be expressed andassayed for VEGF-binding activity, regenerative, pro-angiogenicactivity, promotion of cell proliferation activity, and/or promotioncell migration activity by methods known in the art and/or describedherein to verify that these activities specific to each prom-1 peptideare not abolished by the amino acid substitutions. Variant prom-1peptides have at least 50% of the VEGF-binding activity, regenerative,pro-angiogenic activity, promotion of cell proliferation activity,promotion of cell migration, or neurotrophic/neuroprotective activity ofthe original parent prom-1 peptide. It is contemplated that conservativeamino acid substitution variants of prom-1 peptides as described hereincan have enhanced activity or superior stability relative to the parentprom-1 peptide.

Certain silent or neutral missense mutations can also be made in thenucleic acid encoding a prom-1 peptide by a mutation that does notchange the encoded amino acid sequence or the regenerative and/orpro-angiogenic or anti-angiogenic activities of the encoded prom-1peptide. These types of mutations are useful to optimize codon usagewhich improves recombinant protein expression and production.

Specific site-directed mutagenesis of a nucleic acid encoding a prom-1peptide in a vector can be used to create specific amino acid mutationsand substitutions. Site-directed mutagenesis can be carried out using,e.g., the QUIKCHANGE® site-directed mutagenesis kit from STRATAGENE®according to manufacture's instructions, or by any method known in theart.

Different expression vectors comprising a nucleic acid that encodes aprom-1 peptide or derivative as described herein for the expression andpurification of the recombinant protein produced from a heterologousprotein expression system can be made. Heterologous protein expressionsystems that use host cells selected from, e.g., mammalian, insect,yeast, bacterial, or plant cells are well known to one skilled in theart. The expression vector should have the necessary 5′ upstream and 3′downstream regulatory elements such as promoter sequences, ribosomerecognition and binding TATA box, and 3′ UTR AAUAAA transcriptiontermination sequence for efficient gene transcription and translation inits respective host cell. The expression vector may have additionalsequence such as 6X-histidine (SEQ ID NO: 19), V5, thioredoxin,glutathione-S-transferase, c-Myc, VSV-G, HSV, FLAG, maltose bindingpeptide, metal-binding peptide, HA and “secretion” signals (Honeybeemelittin, α-factor, PHO, Bip), which are incorporated into the expressedrecombinant prom-1 peptide. In addition, there can be enzyme digestionsites incorporated after these sequences to facilitate enzymatic removalof additional sequence after they are not needed. These additionalsequences are useful for the detection of prom-1 peptide expression, forprotein purification by affinity chromatography, enhanced solubility ofthe recombinant protein in the host cytoplasm, for better proteinexpression especially for small peptides and/or for secreting theexpressed recombinant protein out into the culture media, into theperiplasm of the prokaryote bacteria, or to the spheroplast of yeastcells. The expression of recombinant prom-1 peptide can be constitutivein the host cells or it can be induced, e.g., with copper sulfate,sugars such as galactose, methanol, methylamine, thiamine, tetracycline,infection with baculovirus, and (isopropyl-beta-D-thiogalactopyranoside)IPTG, a stable synthetic analog of lactose, depending on the host andvector system chosen.

Recombinant prom-1 peptide can be expressed in a variety of expressionhost cells e.g., bacteria, such as E. coli, yeast, mammalian, insect,and plant cells such as Chlamydomonas, or even from cell-free expressionsystems. From a cloning vector, the nucleic acid can be subcloned into arecombinant expression vector that is appropriate for the expression ofthe prom-1 peptide in mammalian, insect, yeast, bacterial, or plantcells or a cell-free expression system such as a rabbit reticulocyteexpression system. Subcloning can be achieved by PCR cloning,restriction digestion followed by ligation, or recombination reactionsuch as those of the lambda phage-based site-specific recombinationusing the Gateway® LR and BP Clonase™ enzyme mixtures. Subcloning shouldbe unidirectional such that the 5′ ATG start codon of the nucleic acidis downstream of the promoter in the expression vector. Alternatively,when the coding nucleic acid is cloned into pENTR/D-TOPO®,pENTR/SD/D-TOPO® (directional entry vectors), or any of the INVITROGEN'sGateway® Technology pENTR (entry) vectors, the coding nucleic acid canbe transferred into the various Gateway® expression vectors(destination) for protein expression in mammalian cells, E. coli,insects and yeast respectively in one single recombination reaction.Some of the Gateway® destination vectors are designed for theconstructions of baculovirus, adenovirus, adeno-associated virus (AAV),retrovirus, and lentiviruses, which upon infecting their respective hostcells, permit heterologous expression of the prom-1 peptide in the hostcells. Transferring a gene into a destination vector is accomplished injust two steps according to manufacturer's instructions. There areGateway® expression vectors for protein expression in E. coli, insectcells, mammalian cells, and yeast. Following transformation andselection in E. coli, the expression vector is ready to be used forexpression in the appropriate host.

Examples of other expression vectors and host cells are the pET vectors(NOVAGEN), pGEX vectors (Amersham Pharmacia), and pMAL vectors (NewEngland labs. Inc.) for protein expression in E. coli host cells such asBL21, BL21(DE3) and AD494(DE3)pLysS, Rosetta (DE3), and Origami(DE3)(NOVAGEN); the strong CMV promoter-based pcDNA3.1 (INVITROGEN) andpCIneo vectors (PROMEGA) for expression in mammalian cell lines such asCHO, COS, HEK-293, Jurkat, and MCF-7; replication incompetent adenoviralvector vectors pAdeno X, pAd5F35, pLP-Adeno-X-CMV (CLONTECH),pAd/CMV/V5-DEST, pAd-DEST vector (INVITROGEN) for adenovirus-mediatedgene transfer and expression in mammalian cells; pLNCX2, pLXSN, andpLAPSN retrovirus vectors for use with the Retro-X™ system from Clontechfor retroviral-mediated gene transfer and expression in mammalian cells;pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ(INVITROGEN) for lentivirus-mediated gene transfer and expression inmammalian cells; adenovirus-associated virus expression vectors such aspAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (STRATAGENE) foradeno-associated virus-mediated gene transfer and expression inmammalian cells; BACpak6 baculovirus (CLONTECH) and pFastBac™ HT(INVITROGEN) for the expression in Spodopera frugiperda 9 (Sf9) and Sfllinsect cell lines; pMT/BiP/V5-His (INVITROGEN) for the expression inDrosophila Schneider S2 cells; Pichia expression vectors pPICZα, pPICZ,pFLDα and pFLD (INVITROGEN) for expression in Pichia pastoris andvectors pMETα and pMET for expression in P. methanolica; pYES2/GS andpYD1 (INVITROGEN) vectors for expression in yeast Saccharomycescerevisiae. Recent advances in the large scale expression heterologousproteins in Chlamydomonas reinhardtii are described by Griesbeck C. etal. 2006 Mol. Biotechnol. 34:213-33 and Fuhrmann M. 2004, Methods MolMed. 94:191-5. Foreign heterologous coding sequences are inserted intothe genome of the nucleus, chloroplast and mitochondria by homologousrecombination. The chloroplast expression vector p64 carrying theversatile chloroplast selectable marker aminoglycoside adenyltransferase (aadA), which confers resistance to spectinomycin orstreptomycin, can be used to express foreign protein in the chloroplast.The biolistic gene gun method can be used to introduce the vector in thealgae. Upon its entry into chloroplasts, the foreign DNA is releasedfrom the gene gun particles and integrates into the chloroplast genomethrough homologous recombination.

Recombinant protein expression in different host cells can beconstitutive or inducible with inducers such as copper sulfate, orsugars such as galactose, methanol, methylamine, thiamine, tetracycline,or IPTG. After the protein is expressed in the host cells, the hostcells are lysed to liberate the expressed protein for purification.Methods of lysing the various host cells are featured in “SamplePreparation-Tools for Protein Research” EMD Bioscience and in theCurrent Protocols in Protein Sciences (CPPS). A preferred purificationmethod is affinity chromatography such as ion-metal affinitychromatograph using nickel, cobalt, or zinc affinity resins forhistidine-tagged prom-1 peptide. Methods of purifying histidine-taggedrecombinant proteins are described by Clontech using their Talon® cobaltresin and by Novagen in their pET system manual, 10th edition. Anotherpreferred purification strategy is by immuno-affinity chromatography,for example, anti-Myc antibody conjugated resin can be used to affinitypurify Myc-tagged prom-1 peptide. Enzymatic digestion with serineproteases such as thrombin and enterokinase cleave and release theprom-1 peptide from the histidine or Myc tag, releasing the recombinantprom-1 peptide from the affinity resin while the histidine-tags andMyc-tags are left attached to the affinity resin.

Cell-free expression systems are also contemplated. Cell-free expressionsystems offer several advantages over traditional cell-based expressionmethods, including the easy modification of reaction conditions to favorprotein folding, decreased sensitivity to product toxicity andsuitability for high-throughput strategies such as rapid expressionscreening or large amount protein production because of reduced reactionvolumes and process time. The cell-free expression system can useplasmid or linear DNA. Moreover, improvements in translation efficiencyhave resulted in yields that exceed a milligram of protein permilliliter of reaction mix. An example of a cell-free translation systemcapable of producing proteins in high yield is described by Spirin A S.et. al., Science 242:1162 (1988). The method uses a continuous flowdesign of the feeding buffer which contains amino acids, adenosinetriphosphate (ATP), and guanosine triphosphate (GTP) throughout thereaction mixture and a continuous removal of the translated polypeptideproduct. The system uses E. coli lysate to provide the cell-freecontinuous feeding buffer. This continuous flow system is compatiblewith both prokaryotic and eukaryotic expression vectors. As an example,large scale cell-free production of the integral membrane protein EmrEmultidrug transporter is described by Chang G. et. al., Science310:1950-3 (2005).

Other commercially available cell-free expression systems include theExpressway™ Cell-Free Expression Systems (Invitrogen) which utilize anE. coli-based in-vitro system for efficient, coupled transcription andtranslation reactions to produce up to milligram quantities of activerecombinant protein in a tube reaction format; the Rapid TranslationSystem (RTS) (Roche Applied Science) which also uses an E. coli-basedin-vitro system; and the TNT Coupled Reticulocyte Lysate Systems(Promega) which uses a rabbit reticulocyte-based in-vitro system.

Designing Peptide Mimetics

Methods of designing peptide mimetics and screening of functionalpeptide mimetics are well known to those skilled in the art. One basicmethod of designing a molecule which mimics a known protein or peptideis first to identify the active region(s) of the known protein (forexample, in the case of an antibody-antigen interaction, one identifieswhich region(s) of the antibody that permit binding to the antigen), andthen searches for a mimetic which emulates the active region. As anexample, the active region of the prom-1 peptide DRVQRQTTTVVA (SEQ. ID.NO 8) is TTTVVA (SEQ. ID. NO 12). Although the active region of theknown protein is relatively small, it is anticipated that a mimetic willbe smaller (e.g., in molecular weight) than the protein, andcorrespondingly easier and cheaper to synthesize and/or have benefitsregarding stability or other advantageous pharmacokinetic aspects. Sucha mimetic could be used as a convenient substitute for the protein, asan agent for interacting with the target molecule.

For example, Reineke et al. (1999, Nature Biotechnology, 17; 271-275)designed a mimic molecule which mimics a binding site of theinterleukin-10 protein using a large library of short syntheticpeptides, each of which corresponded to a short section of interleukin10. The binding of each of these peptides to the target (in this case anantibody against interleukin-10) was then tested individually by anassay technique, to identify potentially relevant peptides. Phagedisplay libraries of peptides and alanine scanning methods can be used.

Other methods for designing peptide mimetics to a particular peptide orprotein include those described in European Patent EP1206494, theSuperMimic program by Andrean Goede et. al. 2006 BMC Bioinformatics,7:11; and MIMETIC program by W. Campbell et al., 2002, Microbiology andImmunology 46:211-215. The SuperMimic program is designed to identifycompounds that mimic parts of a protein, or positions in proteins thatare suitable for inserting mimetics. The application provides librariesthat contain peptidomimetic building blocks on the one hand and proteinstructures on the other. The search for promising peptidomimetic linkersfor a given peptide is based on the superposition of the peptide withseveral conformers of the mimetic. New synthetic elements or proteinscan be imported and used for searching. The MIMETIC computer program,which generates a series of peptides for interaction with a targetpeptide sequence, is taught by W. Campbell et. al., 2002. In depthdiscussion of the topic is reviewed in “Peptide Mimetic Design with theAid of Computational Chemistry” by James R. Damewood Jr. in Reviews inComputational Chemistry Reviews in Computational Chemistry, January2007, Volume 9 Book Series: Reviews in Computational Chemistry,Editor(s): Kenny B. Lipkowitz, Donald B. BoydPrint ISBN: 9780471186397ISBN: 9780470125861 Published by John Wiley &Sons, Inc.; and in T.Tselios, et. al., Amino Acids, 14: 333-341, 1998.

Methods for preparing libraries containing diverse populations ofpeptides, peptoids and peptidomimetics are well known in the art andvarious libraries are commercially available (see, for example, Eckerand Crooke, Biotechnology 13:351-360 (1995), and Blondelle et al.,Trends Anal. Chem. 14:83-92 (1995), and the references cited therein,each of which is incorporated herein by reference; see, also, Goodmanand Ro, Peptidomimetics for Drug Design, in “Burger's MedicinalChemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons1995), pages 803-861, and Gordon et al., J. Med. Chem. 37:1385-1401(1994), each of which is incorporated herein by reference). One skilledin the art understands that a peptide can be produced in vitro directlyor can be expressed from a nucleic acid, which can be produced in vitro.Methods of synthetic peptide and nucleic acid chemistry are well knownin the art.

A library of peptide molecules also can be produced, for example, byconstructing a cDNA expression library from mRNA collected from a tissueof interest. Methods for producing such libraries are well known in theart (see, for example, Sambrook et. al., Molecular Cloning: A laboratorymanual (Cold Spring Harbor Laboratory Press 1989), which is incorporatedherein by reference). Preferably, a peptide encoded by the cDNA isexpressed on the surface of a cell or a virus containing the cDNA.

Therapeutic/Prophylactic Administration

Pharmaceutical compositions of the present invention can be applied, forexample, topically to a tissue. The composition can be applied as atherapeutically effective amount in admixture with pharmaceuticalcarriers, in the form of topical pharmaceutical compositions. Suchcompositions include solutions, suspensions, lotions, gels, creams,ointments, emulsions, skin patches, etc. All of these dosage forms,along with methods for their preparation, are known in thepharmaceutical and cosmetic art. Harry's Cosmeticology (ChemicalPublishing, 7th ed. 1982); Remington's Pharmaceutical Sciences (MackPublishing Co., 18th ed. 1990). Typically, such topical formulationscontain the active ingredient in a concentration range of 0.1 to 100mg/ml, in admixture with a pharmaceutically acceptable carrier. As usedherein, the terms “pharmaceutically acceptable”, “physiologicallytolerable” and grammatical variations thereof, as they refer tocompositions, carriers, diluents and reagents, are used interchangeablyand represent that the materials are capable of administration to orupon a mammal without the production of undesirable physiologicaleffects such as nausea, dizziness, gastric upset and the like. Apharmaceutically acceptable carrier will not promote the raising of animmune response to a prom-1 peptide with which it is admixed, unless sodesired. The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well understood inthe art and need not be limited based on formulation. For gene therapyusing viral expression, the pharmaceutical compositions can be in theform of an adenovirus, adeno-associated virus, or lentivirus. The dosageranges for gene therapy can be from 1×10⁶ to 10¹⁴ particles perapplication. Other desirable ingredients for use in such preparationsinclude preservatives, co-solvents, viscosity building agents, carriers,etc. The carrier itself or a component dissolved in the carrier may havepalliative or therapeutic properties of its own, including moisturizing,cleansing, or anti-inflammatory/anti-itching properties. Penetrationenhancers may, for example, be surface active agents; certain organicsolvents, such as di-methylsulfoxide and other sulfoxides,dimethyl-acetamide and pyrrolidone; certain amides of heterocyclicamines, glycols (e.g., propylene glycol); propylene carbonate; oleicacid; alkyl amines and derivatives; various cationic, anionic, nonionic,and amphoteric surface active agents; and the like.

Topical administration of a pharmacologically effective amount canutilize transdermal delivery systems well known in the art. An exampleis a dermal patch. Alternatively the biolistic gene gun method ofdelivery may be used. The gene gun is a device for injecting cells withgenetic information, originally designed for plant transformation. Thepayload is an elemental particle of a heavy metal coated with plasmidDNA. This technique is often simply referred to as biolistics. Anotherinstrument that uses biolistics technology is the PDS-1000/He particledelivery system. The isolated prom-1 peptide expression vector, and/orgene therapy viral expression vectors can be coated on minute goldparticles, and these coated particles are “shot” into biological tissuesunder high pressure. An example of the gene gun-based method isdescribed for DNA based vaccination of cattle by Loehr B. I. et al., J.Virol. 2000, 74:6077-86.

In one embodiment, the pharmaceutical compositions described herein canbe administered directly by injection, for example to the affectedtissue, such as organ, muscle or tissue, or wound. A preferredformulation is sterile saline or Lactated Ringer's solution. LactatedRinger's solution is a solution that is isotonic with blood and intendedfor intravenous administration.

In another embodiment, the peptide compositions are administered such asthe agents come into contact with a subject's nervous system. In oneembodiment, the active agents are administered by introduction into thecerebrospinal fluid of the subject. In certain aspects, the peptidecomposition is introduced into a cerebral ventricle, the lumbar area, orthe cistema magna.

In another aspect, the peptide composition is introduced locally, suchas into the site of nerve or cord injury, into a site of pain or neuraldegeneration, or intraocularly to contact neuroretinal cells. In oneembodiment, the peptide composition described herein is administered tothe subject in the period from the time of, for example, an injury tothe CNS up to about 100 hours after the injury has occurred, for examplewithin 24, 12, or 6 hours from the time of injury.

In another embodiment of the invention, the peptide composition isadministered into a subject intrathecally. As used herein, the term“intrathecal administration” is intended to include delivering a peptidecomposition directly into the cerebrospinal fluid of a subject, bytechniques including lateral cerebroventricular injection through aburrhole or cistemal or lumbar puncture or the like (described inLazorthes et al., 1991, and Ommaya A. K., 1984, the contents of whichare incorporated herein by reference). The term “lumbar region” isintended to include the area between the third and fourth lumbar (lowerback) vertebrae. The term “cisterna magna” is intended to include thearea where the skull ends and the spinal cord begins at the back of thehead. The term “cerebral ventricle” is intended to include the cavitiesin the brain that are continuous with the central canal of the spinalcord. Administration of an active compound to any of the above mentionedsites can be achieved by direct injection of the active compoundformulation or by the use of infusion pumps. Implantable or externalpumps and catheter may be used.

An additional means of administration to intracranial tissue involvesapplication of compounds of the invention to the olfactory epithelium,with subsequent transmission to the olfactory bulb and transport to moreproximal portions of the brain. Such administration can be by nebulizedor aerosolized preparations. In a further embodiment, ophthalmic peptidecompositions are used to prevent or reduce damage to retinal and opticnerve head tissues, as well as to enhance functional recovery afterdamage to ocular tissues. Ophthalmic conditions that may be treatedinclude, but are not limited to, retinopathies (including diabeticretinopathy and retrolental fibroplasia), macular degeneration, ocularischemia, and glaucoma. Other conditions to be treated with the methodsdescribed herein include damage associated with injuries to ophthalmictissues, such as ischemia reperfusion injuries, photochemical injuries,and injuries associated with ocular surgery, particularly injuries tothe retina or optic nerve head by exposure to light or surgicalinstruments. The ophthalmic compositions may also be used as an adjunctto ophthalmic surgery, such as by vitreal or subconjunctival injectionfollowing ophthalmic surgery. The peptide compositions may be used foracute treatment of temporary conditions, or may be administeredchronically, especially in the case of degenerative disease. Theophthalmic peptide compositions may also be used prophylactically,especially prior to ocular surgery or noninvasive ophthalmic proceduresor other types of surgery.

In one embodiment, the active compound is administered to a subject foran extended period of time to produce optimum axonal outgrowth.Sustained contact with the peptide composition can be achieved by, forexample, repeated administration of the peptide composition over aperiod of time, such as one week, several weeks, one month or longer.More preferably, the pharmaceutically acceptable formulation used toadminister the active compound provides sustained delivery, such as“slow release” of the active compound to a subject. For example, theformulation may deliver the active peptide composition for at least one,two, three, or four weeks after the pharmaceutically acceptableformulation is administered to the subject. Preferably, a subject to betreated in accordance with the methods described herein is treated withthe active peptide composition for at least 30 days (either by repeatedadministration or by use of a sustained delivery system, or both).

As used herein, the term “sustained delivery” is intended to includecontinual delivery of the active peptide composition in vivo over aperiod of time following administration, preferably at least severaldays, a week, several weeks, one month or longer. Sustained delivery ofthe active compound can be demonstrated by, for example, the continuedtherapeutic effect of the peptide composition over time (such assustained delivery of the agents can be demonstrated by continued axonalgrowth in CNS neurons in a subject). Alternatively, sustained deliveryof the peptide composition may be demonstrated by detecting the presenceof the peptide composition in vivo over time.

Preferred approaches for sustained delivery include use of a polymericcapsule, a minipump to deliver the formulation, a biodegradable implant,or implanted transgenic autologous cells (as described in U.S. Pat. No.6,214,622). Implantable infusion pump systems (such as Infusaid; seesuch as Zierski, J. et al, 1988; Kanoff, R. B., 1994) and osmotic pumps(sold by Alza Corporation) are available in the art. Another mode ofadministration is via an implantable, externally programmable infusionpump. Suitable infusion pump systems and reservoir systems are alsodescribed in U.S. Pat. No. 5,368,562 by Blomquist and U.S. Pat. No.4,731,058 by Doan, developed by Pharmacia Deltec Inc.

In addition to topical therapy it is contemplated that thepharmaceutical compositions described herein can also be administeredsystemically in a pharmaceutical formulation. Systemic routes includebut are not limited to oral, parenteral, nasal inhalation,intratracheal, intrathecal, intracranial, and intrarectal. Thepharmaceutical formulation is preferably a sterile saline or lactatedRinger's solution. For therapeutic applications, the preparationsdescribed herein are administered to a mammal, preferably a human, in apharmaceutically acceptable dosage form, including those that may beadministered to a human intravenously as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-arterial, intrasynovial,intrathecal, oral, or inhalation routes. For these uses, additionalconventional pharmaceutical preparations such as tablets, granules,powders, capsules, and sprays may be preferentially required. In suchformulations further conventional additives such as binding-agents,wetting agents, propellants, lubricants, and stabilizers may also berequired. Vector DNA and/or virus can be entrapped in ‘stabilizedplasmid-lipid particles’ (SPLP) containing the fusogenic lipiddioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol %) ofcationic lipid, and stabilized by a polyethyleneglycol (PEG) coating(Zhang Y. P. et. al. Gene Ther. 1999, 6:1438-47). Other techniques informulating expression vectors and virus as therapeutics are found in“DNA-Pharmaceuticals: Formulation and Delivery in Gene Therapy, DNAVaccination and Immunotherapy” by Martin Schleef (Editor) December 2005,Wiley Publisher, and “Plasmids for Therapy and Vaccination” by MartinSchleef (Editor) May 2001, are incorporated herein as reference. In oneembodiment, the dosage for viral vectors is 1×10⁶ to 10¹⁴ viral vectorparticles per application per patient.

The compositions can be formulated as a sustained release composition.For example, sustained-release means or delivery devices are known inthe art and include, but are not limited to, sustained-release matricessuch as biodegradable matrices or semi-permeable polymer matrices in theform of shaped articles, e.g., films, or microcapsules that compriseprom-1 peptides, expression vectors and/or viral vectors.

A sustained-release matrix, as used herein, is a matrix made ofmaterials, usually polymers, which are degradable by enzymatic oracid/base hydrolysis or by dissolution. Once inserted into the body, thematrix is acted upon by enzymes and body fluids. The sustained-releasematrix desirably is chosen from biocompatible materials such asliposomes, polylactides (polylactic acid), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (co-polymers of lactic acid andglycolic acid) polyanhydrides, poly(ortho)esters, polyproteins,hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fattyacids, phospholipids, polysaccharides, nucleic acids, polyamino acids,amino acids such as phenylalanine, tyrosine, isoleucine,polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.A preferred biodegradable matrix is a matrix of one of eitherpolylactide, polyglycolide, or polylactide co-glycolide (co-polymers oflactic acid and glycolic acid).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers 22:547-556(1983)), poly(2-hydroxyethyl methacrylate) (R. Langer et al., J. BiomedMater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105(1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomally entrapped prom-1 peptides,expression vectors, and nucleic acid constructs. Such liposomes can beprepared by methods known per se: DE 3,218,121; Epstein, et al., Proc.Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. USA 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes areof the small (about 200-800 Angstroms) unilamellar type in which thelipid content is greater than about 30 mol. percent cholesterol, theselected proportion being adjusted for the optimal therapy. Otherbiodegradable polymers and their use are described, for example, indetail in Brem et al. (1991, J. Neurosurg. 74:441-446).

Methods for preparing liposomes and microspheres for administration to apatient are known to those of skill in the art. U.S. Pat. No. 4,789,734,the contents of which are hereby incorporated by reference, describesmethods for encapsulating biological materials in liposomes. A review ofknown methods is provided by G. Gregoriadis, Chapter 14, “Liposomes,”Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press,1979).

Microspheres formed of polymers or proteins are well known to thoseskilled in the art, and can be tailored for passage through thegastrointestinal tract directly into the blood stream. Alternatively,the compound can be incorporated and the microspheres, or composite ofmicrospheres, implanted for slow release over a period of time rangingfrom days to months. See, for example, U.S. Pat. Nos. 4,906,474,4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contentsof which are hereby incorporated by reference.

Preferred micro particles are those prepared from biodegradablepolymers, such as polyglycolide, polylactide and copolymers thereof.Those of skill in the art can readily determine an appropriate carriersystem depending on various factors, including the desired rate of drugrelease and the desired dosage.

In one embodiment, osmotic mini pumps can be used to provide controlledsustained delivery of the pharmaceutical compositions described herein,through cannulae to the site of interest, e.g., directly into a tissueat the site of needing angiogenesis. The pump can be surgicallyimplanted; for example, continuous administration of endostatin, ananti-angiogenesis agent, by intraperitoneally implanted osmotic pump isdescribed in Cancer Res. 2001 Oct. 15; 61(20):7669-74. Therapeuticamounts of prom-1 peptides can also be continually administered by anexternal pump attached to an intravenous needle.

In one embodiment, the formulations are administered via catheterdirectly to the inside of blood vessels. The administration can occur,for example, through holes in the catheter. In those embodiments whereinthe active compounds have a relatively long half life (on the order of 1day to a week or more), the formulations can be included inbiodegradable polymeric hydrogels, such as those disclosed in U.S. Pat.No. 5,410,016 to Hubbell et al. These polymeric hydrogels can bedelivered to the inside of a tissue lumen and the active compoundsreleased over time as the polymer degrades. If desirable, the polymerichydrogels can have microparticles or liposomes which include the activecompound dispersed therein, providing another mechanism for thecontrolled release of the active compounds.

For enteral administration, a composition can be incorporated into aninert carrier in discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compound;as a powder or granules; or a suspension or solution in an aqueousliquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or adraught. Suitable carriers may be starches or sugars and includelubricants, flavorings, binders, and other materials of the same nature.

A tablet can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface activeor dispersing agents. Molded tablets can be made by molding in asuitable machine, a mixture of the powdered active compound with anysuitable carrier.

A syrup or suspension can be made by adding the active compound to aconcentrated, aqueous solution of a sugar, e.g., sucrose, to which canalso be added any accessory ingredients. Such accessory ingredients mayinclude flavoring, an agent to retard crystallization of the sugar or anagent to increase the solubility of any other ingredient, e.g., as apolyhydric alcohol, for example, glycerol or sorbitol.

Formulations for oral administration can be presented with an enhancer.Orally-acceptable absorption enhancers include surfactants such assodium lauryl sulfate, palmitoyl carnitine, Laureth-9,phosphatidylcholine, cyclodextrin and derivatives thereof; bile saltssuch as sodium deoxycholate, sodium taurocholate, sodium glycochlate,and sodium fusidate; chelating agents including EDTA, citric acid andsalicylates; and fatty acids (e.g., oleic acid, lauric acid,acylcarnitines, mono- and diglycerides). Other oral absorption enhancersinclude benzalkonium chloride, benzethonium chloride, CHAPS(3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate),Big-CHAPS(N,N-bis(3-D-gluconamidopropyl)-cholamide), chlorobutanol,octoxynol-9, benzyl alcohol, phenols, cresols, and alkyl alcohols. Anespecially preferred oral absorption enhancer for the present inventionis sodium lauryl sulfate.

Formulations for rectal administration can be presented as a suppositorywith a conventional carrier, e.g., cocoa butter or Witepsol S55(trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

The route of administration, dosage form, and the effective amount varyaccording to the potency of the prom-1 peptides, expression vectors andviral vectors, their physicochemical characteristics, and according tothe treatment location. The selection of proper dosage is well withinthe skill of an ordinarily skilled physician. Topical formulations canbe administered up to four-times a day.

In one embodiment, dosage forms include pharmaceutically acceptablecarriers that are inherently nontoxic and nontherapeutic. Examples ofsuch carriers include ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and polyethylene glycol. Carriers for topical or gel-basedforms of compositions include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,polyethylene glycol and wood wax alcohols. For all administrations,conventional depot forms are suitably used. Such forms include, forexample, microcapsules, nano-capsules, liposomes, plasters, inhalationforms, nose sprays, sublingual tablets, and sustained releasepreparations. For examples of sustained release compositions, see U.S.Pat. No. 3,773,919, EP 58,481A, U.S. Pat. No. 3,887,699, EP 158,277A,Canadian Patent No. 1176565, U. Sidman et al., Biopolymers 22:547 (1983)and R. Langer et al., Chem. Tech. 12:98 (1982). The prom-1 peptides willusually be formulated in such vehicles at a concentration of about 0.1mg/ml to 100 mg/ml and the viral vector should be in the range of 1×10⁶to 10¹⁴ viral vector particles per application per patient.

In one embodiment, other ingredients may be added to pharmaceuticalformulations, including antioxidants, e.g., ascorbic acid; low molecularweight (less than about ten residues) polypeptides, e.g., polyarginineor tripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids, such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose, or dextrins; chelatingagents such as EDTA; and sugar alcohols such as mannitol or sorbitol.

In one embodiment, the pharmaceutical formulation to be used fortherapeutic administration is sterile. Sterility is readily accomplishedby filtration through sterile filtration membranes (e.g., 0.2 micronmembranes). The prom-1 peptide compositions can be stored in lyophilizedform or as an aqueous solution if it is highly stable to thermal andoxidative denaturation. The pH of the prom-1 peptide compositionpreparations typically can be about from 6 to 8.

For therapeutic applications, the appropriate dosage of compositionswill depend upon the type of tissue needing angiogenesis,neuroprotection or other beneficial effect of the prom-1 peptide, theassociated medical conditions to be treated, the severity and course ofthe medical conditions, whether the compositions are administered forpreventative or therapeutic purposes, previous therapy, the patient'sclinical history and response to the compositions and the discretion ofthe attending physician. In addition, in vitro or in vivo assays canoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed will also depend on the route ofadministration, and the seriousness of the condition being treated andshould be decided according to the judgment of the practitioner and eachsubject's circumstances in view of, e.g., published clinical studies.Suitable effective dosage amounts for topical administration of thepeptide compositions described herein range from about 10 micrograms toabout 5 grams applied or administered about every 4 hours, although theyare typically about 500 mg or less per every 4 hours. In one embodimentthe effective dosage for topical administration is about 0.01 mg, 0.5mg, about 1 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg,about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg,about 900 mg, about 1 g, about 1.2 g, about 1.4 g, about 1.6 g, about1.8 g, about 2.0 g, about 2.2 g, about 2.4 g, about 2.6 g, about 2.8 g,about 3.0 g, about 3.2 g, about 3.4 g, about 3.6 g, about 3.8 g, about4.0 g, about 4.2 g, about 4.4 g, about 4.6 g, about 4.8 g, or about 5.0g, every 4 hours. Equivalent dosages may be administered over varioustime periods including, but not limited to, about every 2 hours, aboutevery 6 hours, about every 8 hours, about every 12 hours, about every 24hours, about every 36 hours, about every 48 hours, about every 72 hours,about every week, about every two weeks, about every three weeks, aboutevery month, and about every two months. The effective dosage amountsdescribed herein refer to total amounts administered.

For systemic administration, the dosage ranges are typically from 0.001mg/kg body weight to 5 g/kg body weight. In some embodiments, the dosagerange is from 0.001 mg/kg body weight to 1 g/kg body weight, from 0.001mg/kg body weight to 0.5 g/kg body weight, from 0.001 mg/kg body weightto 0.1 g/kg body weight, from 0.001 mg/kg body weight to 50 mg/kg bodyweight, from 0.001 mg/kg body weight to 25 mg/kg body weight, from 0.001mg/kg body weight to 10 mg/kg body weight, from 0.001 mg/kg body weightto 5 mg/kg body weight, from 0.001 mg/kg body weight to 1 mg/kg bodyweight, from 0.001 mg/kg body weight to 0.1 mg/kg body weight, from0.001 mg/kg body weight to 0.005 mg/kg body weight. Alternatively, insome embodiments the dosage range is from 0.1 g/kg body weight to 5 g/kgbody weight, from 0.5 g/kg body weight to 5 g/kg body weight, from 1g/kg body weight to 5 g/kg body weight, from 1.5 g/kg body weight to 5g/kg body weight, from 2 g/kg body weight to 5 g/kg body weight, from2.5 g/kg body weight to 5 g/kg body weight, from 3 g/kg body weight to 5g/kg body weight, from 3.5 g/kg body weight to 5 g/kg body weight, from4 g/kg body weight to 5 g/kg body weight, from 4.5 g/kg body weight to 5g/kg body weight, from 4.8 g/kg body weight to 5 g/kg body weight. Inone embodiment, the dose range is from 5 μg/kg body weight to 30 μg/kgbody weight. Alternatively, the dose range will be titrated to maintainserum levels between 5 μg/mL and 30 μg/mL.

The compositions comprising prom-1 peptides, expression vectors and/orviral vectors are suitably administered to the patient at one time orover a series of treatments. For purposes herein, a “therapeuticallyeffective amount” of a composition comprising prom-1 peptides, prom-1peptide variants, fusion protein comprising a prom-1 peptide, expressionvector and/or viral vector is an amount that is effective to eitherprevent, reduce the likelihood, lessen the worsening of, alleviate, orcure one or more symptoms or indicia of the treated condition.

Administration of the doses recited above can be repeated for a limitedperiod of time. In some embodiments, the doses are given once a day, ormultiple times a day, for example but not limited to three times a day.In a preferred embodiment, the doses recited above are administereddaily for several weeks or months. The duration of treatment dependsupon the subject's clinical progress and responsiveness to therapy.Continuous, relatively low maintenance doses are contemplated after aninitial higher therapeutic dose.

Therapeutic compositions containing at least one agent can beconventionally administered in a unit dose. The term “unit dose” whenused in reference to a therapeutic composition refers to physicallydiscrete units suitable as unitary dosage for the subject, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requiredphysiologically acceptable diluent, i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. An agent can be targeted by meansof a targeting moiety, such as e.g., an antibody or targeted liposometechnology. In some embodiments, a peptide as described herein can betargeted to tissue- or tumor-specific targets by using bispecificantibodies, for example produced by chemical linkage of an anti-ligandantibody (Ab) and an Ab directed toward a specific target. To avoid thelimitations of chemical conjugates, molecular conjugates of antibodiescan be used for production of recombinant bispecific single-chain Absdirecting ligands and/or chimeric inhibitors at cell surface molecules.The addition of an antibody to a prom-1 peptide permits the agentattached to accumulate additively at the desired target site.Antibody-based or non-antibody-based targeting moieties can be employedto deliver a ligand or the inhibitor to a target site. Preferably, anatural binding agent for an unregulated or disease associated antigenis used for this purpose.

Gene Therapy Using Nucleic Acids Coding for Peptides

The principles of gene therapy are disclosed by Oldham, R. K. (In:Principles of Biotherapy, Raven Press, N.Y., 1987), and similar texts.Disclosures of the methods and uses for gene therapy are provided byBoggs, S. S. (Int. J. Cell Clon. 8:80-96 (1990)); Karson, E. M. (Biol.Reprod. 42:39-49 (1990)); Ledley, F. D., In: Biotechnology, AComprehensive Treatise, volume 7B, Gene Technology, VCH Publishers, Inc.NY, pp 399-458 (1989)), all of which references are incorporated hereinby reference.

In one embodiment, the peptides can be administered to a patient by anyone of several gene therapy techniques known to those of skill in theart. In general, gene therapy can be accomplished by either directtransformation of target cells within the mammalian subject (in vivogene therapy) or transformation of cells in vitro and subsequentimplantation of the transformed cells into the mammalian subject (exvivo gene therapy).

In one embodiment, DNA encoding the peptides can be introduced into thesomatic cells of an animal (particularly mammals including humans) inorder to provide a treatment of a disease or condition that responds tothe peptide composition. Most preferably, viral or retroviral vectorsare employed for this purpose.

Retroviral vectors are a common mode of delivery and in this context areoften retroviruses from which viral genes have been removed or alteredso that viral replication does not occur in cells infected with thevector. Viral replication functions are provided by the use ofretrovirus “packaging” cells that produce the viral proteins requiredfor nucleic acid packaging but that do not produce infectious virus.

Introduction of the retroviral vector DNA into packaging cells resultsin production of virions that carry vector RNA and can infect targetcells, but such that no further virus spread occurs after infection. Todistinguish this process from a natural virus infection where the viruscontinues to replicate and spread, the term transduction rather thaninfection is often used.

In one embodiment, the invention provides a recombinant lentivirus forthe delivery and expression of a peptide in either dividing ornon-dividing mammalian cells. The HIV-1 based lentivirus can effectivelytransduce a broader host range than the Moloney Leukemia Virus(MoMLV)-based retroviral systems. Preparation of the recombinantlentivirus can be achieved using the pLenti4/V5-DEST™, pLenti6/V5-DEST™or pLenti vectors together with ViraPower™ Lentiviral Expression systemsfrom Invitrogen.

Examples of use of lentiviral vectors for gene therapy for e.g.,inherited disorders and various types of cancer, are described in thefollowing references and are hereby incorporated by reference in theirentirety (Klein, C. and Baum, C. (2004). Hematol. J., 5, 103-111;Zufferey, R. et al., (1997). Nat. Biotechnol., 15, 871-875; Morizono, K.et. al. (2005). Nat. Med., 11, 346-352; Di Domenico, C. et al. (2005),Gene therapy for amucopolysaccharidosis type I murine model withlentiviral-IDUA vector. Hum. Gene Ther., 16, 81-90; Kim, E. Y., Hong, Y.B., Lai, Z., Kim, H. J., Cho, Y.-H., Brady, R. O. and Jung, S.-C.(2004). Biochem. Biophys. Res. Comm., 318, 381-390).

Non-retroviral vectors also have been used in genetic therapy. One suchalternative is the adenovirus (Rosenfeld, M. A., et al., Cell 68:143155(1992); Jaffe, H. A. et al., Nature Genetics 1:372-378 (1992);Lemarchand, P. et al., Proc. Natl. Acad. Sci. USA 89:6482-6486 (1992)).Major advantages of adenovirus vectors are their potential to carrylarge segments of DNA (36 Kb genome), a very high titer (10¹¹particles/ml), ability to infect non-replicating cells, and suitabilityfor infecting tissues in situ, especially in the lung. The most strikinguse of this vector so far is to deliver a human cystic fibrosistransmembrane conductance regulator (CFTR) gene by intratrachealinstillation to airway epithelium in cotton rats (Rosenfeld, M. A., etal., Cell 63:143-155 (1992)). Similarly, herpes viruses may also provevaluable for human gene therapy (Wolfe, J. H. et al., Nature Genetics1:379-384 (1992)). Of course, any other suitable viral vector can beused for the genetic therapy for the delivery of prom-1 peptides asdescribed herein.

The viron used for gene therapy can be any viron known in the artincluding but not limited to those derived from adenovirus,adeno-associated virus (AAV), retrovirus, and lentivirus. Recombinantviruses provide a versatile system for gene expression studies andtherapeutic applications.

The recombinant AAV virions described above, including the DNA ofinterest, can be produced using standard methodology, known to those ofskill in the art. The methods generally involve the steps of (1)introducing an AAV vector into a host cell; (2) introducing an AAVhelper construct into the host cell, where the helper construct includesAAV coding regions capable of being expressed in the host cell tocomplement AAV helper functions missing from the AAV vector; (3)introducing one or more helper viruses and/or accessory function vectorsinto the host cell, wherein the helper virus and/or accessory functionvectors provide accessory functions capable of supporting efficientrecombinant AAV (“rAAV”) virion production in the host cell; and (4)culturing the host cell to produce rAAV virions. The AAV vector, AAVhelper construct and the helper virus or accessory function vector(s)can be introduced into the host cell either simultaneously or serially,using standard transfection techniques.

A simplified system for generating recombinant adenoviruses is presentedby He TC. et. al. Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998. Thegene of interest is first cloned into a shuttle vector, e.g.,pAdTrack-CMV. The resultant plasmid is linearized by digesting withrestriction endonuclease Pme I, and subsequently cotransformed into E.coli. BJ5183 cells with an adenoviral backbone plasmid, e.g., pAdEasy-1of Stratagene's ADEASY™ Adenoviral Vector System. Recombinant adenovirusvectors are selected for kanamycin resistance, and recombinationconfirmed by restriction endonuclease analyses. Finally, the linearizedrecombinant plasmid is transfected into adenovirus packaging cell lines,for example HEK 293 cells (E1-transformed human embryonic kidney cells)or 911 (E1-transformed human embryonic retinal cells) (Human GeneTherapy 7:215-222, 1996). Recombinant adenoviruses are generated withinthe HEK 293 cells.

In one embodiment, the invention provides a recombinant adeno-associatedvirus (rAAV) vector for the expression of a peptide, or e.g., a fusionprotein including a peptide as described herein. Using rAAV vectors,genes can be delivered into a wide range of host cells including manydifferent human and non-human cell lines or tissues. Because AAV isnon-pathogenic and does not elicit an immune response, a multitude ofpre-clinical studies have reported excellent safety profiles. rAAVs arecapable of transducing a broad range of cell types and transduction isnot dependent on active host cell division. High titers, >10⁸ viralparticle/ml, are easily obtained in the supernatant and 10¹¹-10¹² viralparticle/ml can be obtained with further concentration. The transgene isintegrated into the host genome, so expression is long term and stable.

The use of alternative AAV serotypes other than AAV-2 (Davidson et al(2000), PNAS 97(7)3428-32; Passini et al (2003), J. Virol77(12):7034-40) has demonstrated different cell tropisms and increasedtransduction capabilities. With respect to brain cancers, for example,the development of novel injection techniques into the brain,specifically convection enhanced delivery (CED; Bobo et al (1994), PNAS91(6):2076-80; Nguyen et al (2001), Neuroreport 12(9):1961-4), hassignificantly enhanced the ability to transduce large areas of the brainwith an AAV vector.

Large scale preparation of AAV vectors is made by a three-plasmidcotransfection of a packaging cell line: AAV vector carrying a DNAcoding sequence for a peptide, AAV RC vector containing AAV rep and capgenes, and adenovirus helper plasmid pDF6, into 50×150 mm plates ofsubconfluent 293 cells. Cells are harvested three days aftertransfection, and viruses are released by three freeze-thaw cycles or bysonication.

AAV vectors are then purified by two different methods depending on theserotype of the vector. AAV2 vector is purified by the single-stepgravity-flow column purification method based on its affinity forheparin (Auricchio, A., et. al., 2001, Human Gene therapy 12; 71-6;Summerford, C. and R. Samulski, 1998, J. Virol. 72:1438-45; Summerford,C. and R. Samulski, 1999, Nat. Med. 5: 587-88). AAV2/1 and AAV2/5vectors are currently purified by three sequential CsCl gradients.

Although local administration will most likely be preferred, a prom-1peptide used in the methods described herein can be deliveredsystemically via in vivo gene therapy. Systemic treatment involvestransfecting target cells with the DNA of interest, i.e., DNA encoding aprom-1 peptide, expressing the coded peptide/protein in that cell, andthe capability of the transformed cell to subsequently secrete themanufactured peptide/protein into the blood.

A variety of methods have been developed to accomplish in vivotransformation including mechanical means (e.g., direct injection ofnucleic acid into target cells or particle bombardment), recombinantviruses, liposomes, and receptor-mediated endocytosis (RME) (forreviews, see Chang et al. 1994 Gastroenterol. 106:1076-84; Morsy et al.1993 JAMA 270:2338-45; and Ledley 1992 J. Pediatr. Gastroenterol. Nutr.14:328-37).

Another gene transfer method for use in humans is the transfer ofplasmid DNA in liposomes directly to human cells in situ (Nabel, E. G.,et al., Science 249:1285-1288 (1990)). Plasmid DNA should be easy tocertify for use in human gene therapy because, unlike retroviralvectors, it can be purified to homogeneity. In addition toliposome-mediated DNA transfer, several other physical DNA transfermethods, such as those targeting the DNA to receptors on cells byconjugating the plasmid DNA to proteins, have shown promise in humangene therapy (Wu, G. Y., et al., J. Biol. Chem., 266:14338-14342 (1991);Curiel, D. T., et al., Proc. Natl. Acad. Sci. USA, 88:8850-8854 (1991)).

Some embodiments of the present invention can be defined as any of thefollowing numbered paragraphs:

1. An isolated prom-1 peptide derived from a prominin-1, the peptidehaving VEGF binding activity.

2. An isolated prom-1 peptide of a prominin-1, the peptide havingpro-angiogenic activity.

3. An isolated prom-1 peptide of a prominin-1, the peptide promoteswound healing.

4. An isolated prom-1 peptide derived from an extracellular domain of aprominin-1, the peptide having pro-angiogenic activity.

5. The isolated prom-1 peptide of any of paragraphs 1-4, wherein theprominin-1 is human prominin-1.

6. The isolated prom-1 peptide of either of paragraphs 4 or 5, whereinthe extracellular domain is one of SEQ. ID. No. 1, 2, or 3.

7. The isolated prom-1 peptide of any one of paragraphs 1-6, wherein thepeptide comprises at least 6 consecutive amino acid residues from SEQ IDNOs: 1, 2, or 3, and wherein the peptide does not include a full-lengthprominin-1.

8. An isolated peptide that is a conservative amino acid substitutionvariant of a peptide of any one of paragraphs 1-7.

9. A peptide that is at least 90% identical to a peptide of any one ofparagraphs 1-8, wherein the peptide has pro-angiogenic activity.

10. An isolated peptide fragment of a prominin-1, the peptide havingpro-angiogenic activity, wherein the peptide consists essentially of apeptide selected from the group consisting of: LCGNSFSGGQPS (SEQ. ID.No. 4); PNIIPVLDEIKS (SEQ. ID. No. 5); LCGVCGYDRHAT (SEQ. ID. No. 6);ITNNTSSVIIEE (SEQ. ID. No. 7); DRVQRQTTTVVA (SEQ. ID. No. 8); andCSFAYDLEAKANSLPPGNLRN (SEQ. ID. No.9), or a conservative amino acidsubstitution variant thereof that substantially retains pro-angiogenicactivity.

11. The isolated peptide of any one of paragraphs 1-10, wherein thepeptide enhances VEGF binding to endothelial cells.

12. The isolated peptide of any one of paragraphs 1-11, wherein thepeptide enhances cell proliferation.

13. The isolated peptide of any one of paragraphs 1-12, wherein thepeptide enhances proliferation of endothelial cells.

14. The isolated peptide of any of paragraphs 1-13, wherein the peptideenhances angiogenesis in the presence of a pro-angiogenic factor.

15. The isolated peptide of any of paragraphs 1-14, wherein the peptideenhances cell migration in the presence of a pro-angiogenic factor.

16. The isolated prom-1 peptide of any one of paragraphs 1-15, whereinthe prom-1 peptide is comprised by a cyclic peptide.

17. The isolated peptide of any of paragraphs 1-15, wherein the peptideis conjugated to a polymer.

18. A fusion protein comprising a peptide of any one of paragraphs 1-17,fused to a heterologous peptide or polypeptide, wherein the fusionprotein is not a full-length prominin-1.

19. A composition comprising a pharmaceutically acceptable carrier andan isolated prom-1 peptide of any one of paragraphs 1-18.

20. A method of promoting cell proliferation in a tissue in needthereof, the method comprising contacting the tissue with a compositionof paragraph 19.

21. A method of promoting angiogenesis in a tissue in need thereof, themethod comprising contacting the tissue with a composition of paragraph19.

22. The method of paragraph 20 or 21, wherein the method is applied inthe context of promoting wound healing, neuronal growth, protection orrepair, tissue repair, fertility promotion, cardiac hypertrophy,treatment of erectile dysfunction, modulation of blood pressure,revascularization after disease or trauma, tissue grafts, or tissueengineered constructs.

23. A method of promoting wound healing, the method comprisingcontacting the wound with an isolated prom-1 peptide, cyclic peptide, orfusion protein of any one of paragraphs 1-19, whereby wound healing isenhanced relative to wound healing in the absence of the peptide orfusion protein.

24. A method of promoting neuroprotection or neural regenerationcomprising contacting a neuronal cell with an isolated prom-1 peptide ofa prominin-1.

25. The method of paragraph 22 wherein the prom-1 peptide of aprominin-1 comprises sequence found in an extracellular domain of theprominin-1.

26. The method of paragraph 22 or paragraph 23 wherein the prominin-1 ishuman prominin-1.

27. The method of paragraph 23 or paragraph 24, wherein theextracellular domain is one of SEQ. ID. No. 1, 2, or 3.

28. The method of any one of paragraphs 22-25, wherein the prom-1peptide comprises at least 6 consecutive amino acid residues of thefull-length prom-1, and wherein the peptide does not include afull-length prominin-1.

29. The method of any one of paragraphs 22-26 wherein the prom-1 peptidecomprises a conservative amino acid substitution relative to thecorresponding wild-type human prominin-1 sequence.

30. The method of any one of paragraphs 22-27 wherein the prom-1 peptideis a conservative amino acid substitution variant of a peptide of SEQ IDNO: 8.

31. The method of any one of paragraphs 22-28 wherein the prom-1 peptideis comprised by a cyclic peptide.

32. The method of any one of paragraphs 22-29 wherein the prom-1 peptideis comprised by a heterologous fusion polypeptide.

33. The method of any one of paragraphs 22-30 wherein the prom-1 peptideis conjugated to a polymer.

34. The method of any one of paragraphs 22-31 wherein the prom-1 peptideconsists essentially of a peptide of SEQ ID NO: 8.

35. The method of any one of paragraphs 22-32 wherein the prom-1 peptideconsists of SEQ ID NO: 8.

36. The method of any one of paragraphs 22-33 wherein the contactingcomprises administering a pharmaceutical composition comprising theprom-1 peptide and a pharmaceutically acceptable carrier to anindividual in need of neuroprotection.

37. The method of any one of paragraphs 22-34 wherein the contactingprevents or delays neuronal cell death relative to neuronal cell deathoccurring in the absence of the contacting.

38. The method of any one of paragraphs 22-35 wherein the contactingpromotes nerve regeneration.

39. A cyclic peptide of prominin-1 comprising the formula ofCX(DRVQRQTTTVVA)ZC or ACX(DRVQRQTTTVVA)ZC, wherein X or Z are eachindependently 0-20 amino acids.

40. The cyclic peptide of paragraph 39 comprising a sequence selectedfrom the group consisting of: ACGG(DRVQRQTTTVVA)GGC (SEQ ID NO: 15),ACGG(DRVQRQTTTVVA)GGGGGGC (SEQ ID NO: 16), and CGGGGGG(DRVQRQTTTVVA)GGCA(SEQ ID NO: 17).

41. The cyclic peptide of paragraph 37 or 38, wherein the peptideenhances VEGF binding to endothelial cells.

42. The cyclic peptide of any one of paragraphs 37-39, wherein thepeptide enhances cell proliferation.

43. The cyclic peptide of any one of paragraphs 37-40, wherein thepeptide enhances proliferation of endothelial cells.

44. The cyclic peptide of any one of paragraphs 37-41, wherein thepeptide enhances angiogenesis in the presence of a pro-angiogenicfactor.

45. The cyclic peptide of any one of paragraphs 37-42, wherein thepeptide enhances cell migration in the presence of a pro-angiogenicfactor.

46. The cyclic peptide of any one of paragraphs 37-43, wherein thepeptide promotes wound healing.

47. The cyclic peptide of any one of paragraphs 37-43 wherein theprominin-1 is human prominin-1.

48. An isolated prom-1 peptide of any one of claim 1-19 or 40-47,wherein said peptide stimulates neuronal growth.

49. An isolated prom-1 peptide of a prominin-1, said peptide havingneuronal growth stimulatory activity.

50. A method for stimulating neuron growth, the method comprisingcontacting a neuron with a peptide of any one of claim 1-19 or 40-49.

This invention is further illustrated by the following examples whichshould not be construed as limiting.

EXAMPLES Example 1 Seven 12-Mer Peptides Derived from Prominin-1Sequence Bind VEGF

Minimum epitope assignment was based on immunostaining of ABIMED spotpeptide arrays prepared at the MIT Biopolymers Facility (FIG. 2). Eachspot comprised a 12-mer contiguous peptide, and depending on the numberof residues in the antigen of interest, 3-residue offset was used tocover the entire antigen sequence. For 3-residue offset, spot 1 containssequence 1-12, spot 2 contains sequence 3-15, spot 3 contains sequence6-18, etc. The Cellulose-bound peptide membrane was preincubated withT-TBS blocking buffer (TBS, pH 8.0/0.05% Tween 20 in the presence ofblocking reagent; Roche Diagnostics chemiluminescence detection kit1500694). Subsequently, the peptide array was incubated with hVEGF at afinal concentration of 1.0 μg/ml for 2 h in T-TBS blocking buffer. Afterwashing three times for 10 min with T-TBS the anti-hVEGF antibody(Quantum Biotechnologies, Montreal), was added to a final concentrationof 11 g/ml in T-TBS blocking buffer for 1 h followed by washing threetimes for 10 min with T-TBS. Finally, the arrays were incubated with asecond anti-mouse IgG peroxidase-labeled antibody (catalog no. A5906,Sigma), which was applied at a concentration of 11 g/ml in T-TBSblocking buffer for 1 h, followed by washing three times for 10 min withT-TBS. Analysis of peptide-bound VEGF-antibody complexes was done byusing a chemiluminescence substrate. Binding of the detection antibodyto the peptides was excluded by control incubations with anti-mouse IgGperoxidase-labeled antibody alone (data not shown). The seven highlyreacted peptides were commercially synthesized (Genescript Co., NJ)(Table 1) and confirmed for binding to VEGF-A by ELISA and dot blot(data not shown).

Example 2 Peptide 237 Derived from the Extracellular Domain of PromininDramatically Increases VEGF Binding to Endothelial and B16-F10 MelanomaCells

In order to characterize the effect of the peptides on VEGF binding toendothelial and melanoma cells, the cells were incubated with variouspeptides (720 μg/ml) in the presence of h25-VEGF (12 ng/ml) (FIG. 3).Peptide #237 increased VEGF binding to endothelial as well to melanomacells. 10000 cells were incubated in binding buffer containing 20 mMHepes, 0.1% BSA and I¹²⁵-VEGF (12 ng/ml) for 3 h on ice. Following 3washings, the radioactive levels were determined by gamma counter. Anincrease of more than 25 times in I¹²⁵-VEGF binding was observed whenpeptide #237 was added to both kinds of cells. All other peptides had noeffect on VEGF binding.

Example 3 Determination of the Essential Amino Acids for VEGF Binding inPeptide #237

To characterize which are the essential amino acids within the #237peptide that are crucial to accelerate VEGF binding to the cells, threeshorter peptides were designed as presented at Table 2. The cells weretreated with the smaller peptides in the same way as described in FIG.3. 10000 cells were incubated in binding buffer containing 20 mM Hepes,0.1% BSA, I¹²⁵-VEGF (12 ng/ml) and 100 μg/ml of each peptide, for 3 h onice. Following 3 washings with binding buffer, the radioactive levelswere determined using a gamma counter. It is concluded that the first6-mer peptide (#237A) is not crucial to enhance VEGF binding to cellswhereas the second 6-mer (#237B) shows a partial increase in VEGFbinding to endothelial cells and better binding to melanoma cells. Inaddition, the amino acids valine and alanine, located in the C terminusof the peptide, were found to be crucial in the VEGF binding process.When these two amino acids were removed from the original peptide aspresented by #237C, the VEGF binding was essentially eliminated.

TABLE 2 #237 peptide fragments #237 DRVQRQTTTVVA SEQ ID NO: 8 A DRVQRQSEQ ID NO: 13 B       TTTVVA SEQ ID NO: 12 C   VQRQTTTV SEQ ID NO: 14

In addition, a series of peptides derived from peptide #237 weredesigned to test the sequence dependency of the peptide to enhance VEGFbinding to endothelial cells. The peptides were synthesized bysequentially deleting an amino acid from either the N-terminus orC-terminus and adding to the opposite terminus the next amino acid inthe prominin-1 sequence. Essentially, the peptide sequence is shifted ineither direction by one amino acid in the promonin-1 sequence at a time.Each of these peptides were tested for VEGF binding to endothelial cellsand compared to peptide #237 at varying dilutions. The results areindicated herein in FIG. 9.

Example 4 Extracellular Fragments of Prominin-1 Affect Endothelial andMelanoma Cell Proliferation

Cell proliferation was assessed using an assay based on the cleavage ofthe tetrazolium salt WST-1 to formazan by cellular mitochondrialdehydrogenases. Aliquot of 50,000 micro-vessel endothelial cells (FIG.5A) or F10-B16 melanoma cells (FIG. 5B) were added to each well of a96-well plate in EGM medium containing 10% fetal bovine serum. Aftercells had attached to the 96-well tray, the cells were washed, and highserum medium was replaced with starvation medium overnight. All wellswere rinsed with phosphate-buffered saline. Negative control wellsreceived starvation medium, and positive control wells received fullmedium. The cells were treated with the different peptides (100 ug/ml)to determine their effect on cell proliferation. Cells were allowed toincubate for 24 h in the presence of the respective peptide. At thistime, the WST-1 reagent, was applied for 4 h to measure cellproliferation. The plates were read on OD=450 nm, and data werepresented as a percentage of negative control proliferation, with p<0.05being significant. Expansion in the number of viable cells results in anincrease in the overall activity of the mitochondrial dehydrogenases inthe wells. As shown in FIG. 5, human umbilical vein endothelial cell(HUVEC) and B16-F10 melanoma cell proliferation were significantlyincreased by incubation with cellular fragments of Prominin-1 (p<0.05).

Example 5 Prominin Fragment (#237) Dramatically Increases AngiogenesisIn Vivo when Added to the VEGF Pellet During Corneal Micropocket Assay

In order to evaluate the modulating effect of peptide #237 on theangiogenesis process, a corneal micropocket assay was performed. Twokinds of pellets were created, both containing 160 ng carrier-freerecombinant human VEGF 165 (R&D Systems, Minneapolis, Minn.), one ofwhich contains 1.3 ug of #237 peptide. The pellets were implanted intomicropockets created in the cornea of two groups of anesthetized mice(n=4). Through the use of standardized slow-release pellets, apredictable angiogenic response is generated over the course of 5 daysand then quantified. The area of neovascularization was calculated asvessel area, which is calculated as the product of vessel lengthmeasured from the limbus and clock hours around the cornea, using thefollowing equation: vessel area (mm²)=[clock hours×vessel length(mm)×0.2 mm]. An increase of 53% was observed in the vessel density inthe treated eyes (pellet with peptide #237) versus the control (pelletwithout peptide #237) (FIG. 6). The in vivo experiment confirms that thepeptide stimulates cell proliferation, and is therefore a good candidateto induce angiogenesis.

Example 6 Prominin Fragment (#237) Increases Endothelial Cell MigrationIn Vivo when Combined with VEGF as Tested in the Matrigel Assay

Two groups of 8-week-old C57bl mice were anaesthetized and injectedsubcutaneously with either 0.5 ml ice-cold Matrigel supplemented with500 ng VEGF (0.5 μg/ml), or with Matrigel containing VEGF and Promininfragment-#237 (180 μg). On day 6, animals were sacrificed andfluorescence-activated cell sorting (FACS) analysis was used fordetermination of the matrigel liberated cells. In order to distinguishthe endothelial cells from hematopoietic cells, the cells were incubatedwith two antibodies CD31-PE and CD45-APC which are specific toendothelial cells and hematopoietic cells respectively. The left upperpanel reflects the number of the endothelial cells which are the cellsthat builds vessels. As shown in FIG. 7, six times more endothelialcells (0.42% versus 0.07%) (upper left panel) were observed when the 180ug of #237 peptide was added to the VEGF. This observation confirms that#237 is a potent angiogenic factor which has a synergistic effect onendothelial cell migration induced by VEGF.

Example 7 Prominin Fragment (#237) Increases Neovascularization in aWound Healing Model

Microvascular in-growth into damaged tissue is an essential component ofthe normal healing process. In fact, wound therapy is often aimed atpromoting neovascularization. The model consists of wounding the dorsalaspect of the ear of a nude mouse. A circular punch was used to create astandardized wound on a nude mouse ear. Constant diameter of wound isgiven by the size of the punch (2.25 mm), and the ears were treateddaily with plain matrigel (FIG. 8A) or matrigel containing peptide #237(180 μg) (FIG. 8B) for 5 days. Matrigel solution had minimal effect onneovascularization, but in contrast, the matrigel containing peptide#237 significantly increased the neovascularization around the ear woundsite (×4). In order to evaluate the wound neovascularization, the micewere inoculated by dextran-FITC which specifically labeled theendothelial cells, immediately after anesthetizing them. The ear/woundwas observed and as shown in FIG. 8, the angiogenesis around thecircular wound among the treated mice (the #237 peptide) was greaterthan among the untreated mice.

Example 8 Circularized Peptides of Prominin-1

Provided herein are exemplary circular peptides of prominin-1 that arecontemplated for use with the methods and compositions described herein.The amino acids GG are added prior to the cysteine residue to permitdisulfide bridge formation.

Exemplary sequences for circular peptides of peptide #237 include, butare not limited to, ACGGDRVQRQTTTVVAGGC (SEQ ID NO: 15);ACGGDRVQRQTTTVVAGGGGGGC (SEQ ID NO: 16); and CGGGGGGDRVQRQTTTVVAGGCA(SEQ ID NO: 17).

Further cyclic peptides for e.g., peptide #237 can be designed using thefollowing exemplary formulas. These formulas permit a peptide to beconverted into a cyclic peptide by the formation of a disulfide bondbetween the two cysteines. Exemplary formulas for designing cyclicpeptides of peptide #237 are shown below:

CX(DRVQRQTTTVVA)ZC, (SEQ ID NO: 37) ACX(DRVQRQTTTVVA)ZC, (SEQ ID NO: 38)wherein X or Z are each independently 0-20 amino acids used as spacers.In the specific examples for peptide 237 shown above (SEQ ID NO: 15; SEQID NO: 16; SEQ ID NO: 17) these spacer amino acids are G.

For non-237 based sequences, examples of formulas based upon otheractive linear peptide sequences that can then be made into cyclicpeptides by the formation of a disulfide bond between the two cysteinesare below:

CX(active linear peptide sequence)ZC,A CX(active linear peptide sequence)ZC,CX(active linear peptide sequence)ZCA,wherein X or Z are each independently 0-20 amino acids used as spacers.

Example 9 Peptide #237 Promotes Wound Healing

In addition to increasing angiogenesis, peptide #237 also accelerateswound healing. In order to evaluate the effect of the peptide on woundhealing, ears of five mice were wounded using a circular punch, whichcreates a wound measuring 1 mm. The ears of wounded mice were treateddaily with either MATRIGEL or MATRIGEL containing #237 peptide for 14days. As shown in FIG. 10, the wound area of the treated mice wassignificantly smaller than among the untreated mice on day 14,indicating that #237 promotes wound healing in a mouse model.

Example 10 Peptide #237 Promotes Neurite Outgrowth

Primary cortical neuronal cells (2×10⁴ per well) were plated onpoly-L-lysine coated 24 well dishes and treated with either scrambled#237 or peptide #237 (0.25 μg/μL). Cells were then cultured for 2 days.

Neurite outgrowth was evaluated by counting the number of neuritesextended from the same number of cells and at the same area. As shown inFIG. 11, peptide #237 dramatically induces branched and longer neuritescompared to cells treated with a scrambled peptide. Without wishing tobe bound by theory, these data indicate that peptide #237 stimulatesneuronal growth and regeneration.

Peptide 237 increases neurite outgrowth in terminally differentiatedcortical neurons. The number of neurite arborizations in thesepostmitotic cells was estimated using light microscopy by manuallycounting the number of projections in a given field containingapproximately equivalent number of cells. Quantification of neuriteoutgrowth was also performed by fluorescent staining with aneuron-specific marker (i.e., neurofilament) followed by automated,computational analysis (data not shown).

Example 11 Peptide #237 Improves Blood Flow in Ischemic Limbs in a Mouse

The therapeutic activity on ischemic tissues in mice of peptide #237(DRVQRQTTTVVA) (SEQ. ID. NO: 8) was demonstrated using a mouse hind limbischemia model in which ischemia was induced by femoral artery ligationin one leg. Mice were ligated in one of their femoral arteries (theright limb) to simulate hind limb ischemia. The blood flow in mice wasanalyzed by machine for each mouse before and immediately after theprocedure to evaluate if the femoral was ligated properly. Blood flowwas compared with the non-operated leg by a laser Doppler imager.Peptide #237 was administered intraperitoneal immediately followingfemoral artery occlusion significantly improves limb perfusion, withinthree to six days. The ischemic limb is seen as the limb on left side inthe laser Doppler images.

FIG. 12 shows a representative evaluation of the ischemic (left side ofeach image) and non-ischemic (right side of each image) hind limbs,immediately after, and on days 4 and 14 after surgery. In FIG. 12, thewhite areas indicate normal perfusion with normal blood circulation andblack areas indicate no blood flow in the ischemic left hind limb. Miceadministered with peptide #237 showed increased blood flow recoverycompared to saline-treated mice. Dark shaded areas within the whiteareas located at the distal ends of the limbs indicate extremely goodblood flow.

The blood flow of the ischemic hind limb is expressed as the ratiobetween the perfusion of the ischemic limb and the uninjured limb inFIG. 13. On average, the peptide #237-treated mice showed about 30-40%increase perfusion compared to saline-treated mice.

Example 12 Peptide #237 Improves Bone Repair in Calvaria Critical SizeDefect Experiment

In this study, the inventors examined the ability of peptide #237 toenhance repair of a critical size defect in a rat calvaria critical sizedefect experiment. Critical size calvarial defects (5-mm diameter) werecreated in rats and locally treated with saline (control) or peptide#237 for 28 days (100 μg/mice/5 days). After 28 days, analysis of boneregeneration was determined by soft x-ray.

FIG. 14 shows the extent of bone regeneration in the mice administeredwith peptide #237 compared with control mice that were treated withsaline only. The edges of the calvaria on the animal treated withpeptide #237 have completely sealed and covered the aperture while thoseof the control animal treated with saline are still very distinct after28 days.

Example 10 Alanine Substituted Peptide #237 at Position 5 (Ala 5)Enhanced VEGF Binding by Endothelial Cells

The inventors also investigated which amino acid residues in peptide#237 are important for the various angiogenic and VEGF related bindingactivity described herein. To achieve this, the inventors singly changedeach of the 12 amino acid residues in peptide #237 to alanine andperformed experiments examining the effect of the alanine substitutionat various positions on endothelial cell binding to VEGF. A total of 12alanine substituted peptides were made and tested: Ala-1, Ala-2, Ala-3,Ala-4, Ala-5, Ala-6, Ala-7, Ala-8, Ala-9, Ala-10, Ala-11, Ala-12, (SEQ.ID. NOS. 39-50) wherein the number indicate the position where thealanine substitution occurred. (Note that the amino acid at position 12of peptide #237 was changed to glycine in Ala-12). In addition, a “flip#237” peptide having the sequence AAVVTTTQRQVRD (SEQ. ID. NO. 53) wasalso made and studied.

FIGS. 15A and 15B show that alanine substitutions showed a markedincrease in activity when the substitution was made at position 5. Ala-5peptide has the arginine at position 5 replaced by alanine. The abilityof endothelial cells to bind VEGF was increased by more than twicecompared to the treatment with the original #237.

The references cited herein and throughout the specification areincorporated herein by reference.

TABLE 1 Stimulate VEGF Endo- ↑angio- ↑Endo- Peptide Amino acid Seq.binding thelial genesis thelial Sequence Sequence of ID. VEGF in otherCell in cell Number peptide No. binding cells growth VEGF migrate   #3LCGNSFSGGQPS 4 + −(1)  +/+  #42 PNIIPVLDEIKS 5 + −(1)  +/+  #77LCGVCGYDRHAT 6 + −(1) +/ #122 ITNNTSSVIIEE 7 + −(1) #237 DRVQRQTTTVVA8 + +(2)  +/+ + + #640 CSFAYDLEAKANSLPPGNL 9 + −(1)  +/+ RN

1. An isolated prom-1 peptide derived from a prominin-1, wherein said peptide has regenerative activity, and wherein the peptide does not include a full-length prominin-1.
 2. The isolated peptide from claim 1, wherein said peptide's regenerative activity is promoting angiogenesis.
 3. The isolated peptide from claim 1, wherein said peptide's regenerative activity is promoting neuronal growth.
 4. The isolated peptide from claim 1, wherein said peptide's regenerative activity is promoting wound healing in tissues and/or bone.
 5. The isolated peptide from claim 1, wherein said peptide is derived from an extracellular domain of a prominin-1.
 6. The isolated prom-1 peptide from claim 1, wherein the peptide consists essentially of a peptide selected from the group consisting of: LCGNSFSGGQPS (SEQ. ID. No. 4); PNIIPVLDEIKS (SEQ. ID. No. 5); LCGVCGYDRHAT (SEQ. ID. No. 6); ITNNTSSVIIEE (SEQ. ID. No. 7); DRVQRQTTTVVA (SEQ. ID. No. 8); and CSFAYDLEAKANSLPPGNLRN (SEQ. ID. No.9).
 7. The isolated prom-1 peptide of claim 1, wherein the peptide comprises at least 6 contiguous amino acid residues, and wherein the peptide does not include a full-length prominin-1.
 8. An isolated peptide that is a conservative amino acid substitution variant of a peptide of claim
 1. 9. A peptide that is at least 90% identical to a peptide of claim 1, wherein said peptide has regenerative activity.
 10. A peptide of claim 1, wherein said prom-1 peptide is constructed into a cyclic peptide.
 11. The peptide of claim 1, wherein the cyclic peptide comprises a formula of CX(DRVQBQTTTVVA)ZC (SEQ ID NO: 51) or ACX(DRVQBQTTTVVA)ZC (SEQ ID NO: 52), wherein X or Z are each independently 0-20 amino acids, and wherein B is any one of the naturally occurring amino acid or a derivative thereof.
 12. The peptide of claim 11, wherein the glutamine (Q) at position 6 of the core (DRVQBQTTTVVA) (SEQ ID NO: 54) of the cyclic peptide is substituted with any one of the known 20 amino acids other that glutamine.
 13. A peptide of claim 1, wherein the cyclic peptide is selected from the group consisting of: ACGG(DRVQ[[B]]RQTTTVVA)GGC (SEQ ID NO: 15), ACGG(DRVQ[[B]]RQTTTVVA)GGGGGGC (SEQ ID NO: 16), and CGGGGGG(DRVQ[[B]]RQTTTVVA)GGCA (SEQ ID NO: 17).
 14. A peptide of claim 1 with a formula of X(DRVQBQTTTVVA)Z (SEQ ID NO: 59) or X(DRVQBQTTTVVA)Z (SEQ ID NO: 60), wherein X or Z are each independently 0-20 amino acids, and wherein B is any one of the naturally occurring amino acid or a derivative thereof.
 15. A peptide of claim 13 which is a conservative amino acid substitution variant or a 90% homologous variant thereof that substantially retains regenerative activity.
 16. The isolated prom-1 peptide of claim 1, wherein the peptide is conjugated to a polymer.
 17. A fusion protein comprising a peptide of claim 1, fused to a heterologous peptide or polypeptide, wherein the fusion protein is not a full-length prominin-1.
 18. A composition comprising a pharmaceutically acceptable carrier and an isolated prom-1 peptide of claim 1 or fusion protein of claim
 16. 19. A method of promoting regeneration in a tissue in need thereof, the method comprising contacting said tissue with a composition of claim
 17. 20. The method of claim 18, wherein the method is applied in the context of promoting wound healing, neuronal growth, protection or repair, tissue repair, bone repair, fertility promotion, cardiac hypertrophy, treatment of erectile dysfunction, modulation of blood pressure, revascularization after disease or trauma, tissue grafts, or tissue engineered constructs.
 21. A method of promoting neuroprotection or neural regeneration to an individual in need of neuroprotection, the method comprising contacting a neuronal cell with a composition of claim 18, wherein said contacting prevents or delays neuronal cell death relative to neuronal cell death occurring in the absence of said contacting, or wherein said contacting promotes nerve regeneration by stimulating neuronal growth. 